Engineered poypeptides having enhanced duration of action and reduced immunogenicity

ABSTRACT

Compounds are provided having inter alia good duration of action, high potency and/or convenient dosing regimens including once weekly administration. The compounds are engineered polypeptides which incorporate an albumin binding domain in combination with one or more biologically active polypeptides. Also provided are pharmaceutical compositions and methods of treatment for diseases and disorders including lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer&#39;s disease, leptin deficiency, fatty liver disease or diabetes (including type I and type II). Additional diseases and disorders which can be treated by the compounds and methods described herein include nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X and Huntington&#39;s Disease.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 3, 2012, isnamed 1320USPR.txt and is 450,377 bytes in size.

BACKGROUND OF THE INVENTION

The present application relates to compounds having good duration ofaction, high potency and/or convenient dosing regimens, and method ofuse thereof. There are provided herein engineered polypeptides whichincorporate an albumin binding domain in combination with a biologicallyactive peptide. Without wishing to be bound by any theory, it isbelieved that because the engineered polypeptides described herein canbind albumin, the compounds can be sequestered (e.g., bound to albumin)while in the circulation leading to increased duration of action, duefor example to decreased renal clearance and/or degradation. Diseasesamenable to such treatment include lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes(including type I and type II), nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X andHuntington's Disease, or combinations thereof.

There remains a need to develop polypeptides useful in the abovedescribed metabolic diseases, conditions and disorders. Accordingly, itis an object of the present invention to provide engineered polypeptideswith extended half-lives useful to treat the above conditions andmethods for producing and using them.

Each patent, patent application, and publication cited herein is herebyincorporated herein by reference in its entirety and for all purposes.

BRIEF SUMMARY OF THE INVENTION

There are provided engineered polypeptide compounds having bindingaffinity for albumin and an additional therapeutic utility. Thecompounds are engineered polypeptides which include an albumin bindingdomain (ABD) polypeptide capable of binding albumin and a hormone domain(HD) polypeptide, which HD polypeptides can be biologically active andcan elicit a beneficial biological response, in covalent linkage withthe ABD. Any of the ABD or HD polypeptides described herein can beoptionally covalently bonded in the engineered polypeptide through alinker L, for example L1 as described herein. Without wishing to bebound by any theory, it is believed that because the engineeredpolypeptides described herein can bind albumin, the compounds can besequestered in a subject leading to increased duration of action in thesubject.

In a first aspect, there is provided an engineered polypeptide asdescribed herein. The engineered polypeptide includes an albumin bindingdomain polypeptide (ABD) and a hormone domain (HD1). The hormone domainincludes a polypeptide which is a leptin, an analog of a leptin or anactive fragment thereof.

In another aspect, there is provided a method for treating a disease ordisorder in a subject in need of treatment. The method includesadministering an engineered polypeptide as described herein to thesubject.

In yet another aspect, there is provided a pharmaceutical compositionwhich includes an engineered polypeptide compound described herein incombination with a pharmaceutically acceptable excipient.

In yet another aspect are polynucleotides encoding the engineeredpolypeptide and their intermediates, expression vectors bearing suchpolynucleotides, host cells expressing such polynucleotides, and meansfor their expression, synthesis, post-translational modification andisolation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a listing of the amino acid sequences of examples of albuminbinding polypeptides (SEQ ID NO:301-452, SEQ ID NO:454-463, SEQ IDNO:500-593) useful in the engineered polypeptides disclosed herein, theGA3 domain from protein G of Streptococcus strain G148 (SEQ ID NO:453)extended by a N-terminal glycine residue and an albumin bindingpolypeptide derived from G148-GA3 as previously described by Jonsson etal (Protein Eng. Design & Selection, 2008, 21:515-527; SEQ ID NO:454).

FIG. 2 shows the result of binding analysis performed in a Biacoreinstrument for investigating the binding of the albumin bindingpolypeptide PEP07912 (SEQ ID NO: 456) to human serum albumin. Threedifferent concentrations of purified protein (40 nM, fat gray line; 10nM, black line; and 2.5 nM, gray line) were injected over a surface with955 RU of immobilized human serum albumin.

FIGS. 3A-C show the result of binding analysis performed by ELISA forinvestigating the binding of the albumin binding polypeptides PEP07913(SEQ ID NO:453), PEP06923 (SEQ ID NO:454), PEP07271 (SEQ ID NO:455),PEP07912 (SEQ ID NO:457), PEP07554 (SEQ ID NO:456), PEP07914 (SEQ IDNO:458), PEP07968 (i.e. DOTA conjugated to PEP07911 (SEQ ID NO:459)) andPEP07844 (SEQ ID NO:461), to IgG molecules present in 126 individualnormal human sera, where A) shows the average OD-value, B) shows thepercentage of negative sera (defined as OD<0.15), and C) shows thepercentage of positive sera (defined as OD>1.0).

FIGS. 4A-C are diagrams showing an immunogenicity assessment of albuminbinding polypeptides PEP07913 (SEQ ID NO:453), PEP07912 (SEQ ID NO:457),PEP07914 (SEQ ID NO:458) and PEP07968 (i.e. DOTA conjugated to PEP07911(SEQ ID NO:459)) in a CD3+ CD4+ T cell proliferation assay. A) shows thenumber of individuals responding to the albumin binding polypeptidescompared to recombinant human albumin in a cohort of 52 Caucasiandonors. B) shows the average stimulation indices (SI) for PEP07913,PEP07912, PEP07914 and PEP07968 compared to the negative controlcontaining recombinant human albumin. C) shows the number of respondingindividuals against all proteins in the study as compared to the buffercontrol.

FIG. 5 depicts the leptin functional activity generated by Compound 2 inthe presence of albumin, as described in Example 7.

FIGS. 6A-6B depict the effects of a single administration of theindicated engineered polypeptides described herein on food intake andchange in body weight (% vehicle-corrected) upon administration to ratsas described in Example 8. FIG. 6A: food intake. FIG. 6B: change in bodyweight (% vehicle-corrected).

FIGS. 7A-7B depict the effects of a single administration of theindicated engineered polypeptides described herein on food intake andchange in body weight (% vehicle-corrected) upon administration to ratsas described in Example 9. FIG. 7A: food intake. FIG. 7B: change in bodyweight (% vehicle-corrected).

FIG. 8 depict the effects of a single administration of the indicatedengineered polypeptides described herein on change in body weight (%vehicle-corrected) upon administration to rats as described in Example10.

FIG. 9 depicts the effects of a single administration of the indicatedengineered polypeptides described herein on change in body weight (%vehicle-corrected) upon administration to rats as described in Example11.

FIG. 10 is a graph depicting the effect on body weight of administrationof leptin (125 μg/kg/day) and amylin (1500 μg/kg/day), either alone orin combination, in two groups of rats: one group of very obese rats andanother group that was calorie restricted down to the range of moderateobesity.

FIG. 11 is a graph depicting an effect on body weight of administrationof Compound 64 (120 nmol/kg) and PEG-rat amylin(Des-Lys1-[Lys26(mPEG40K)]-Rat Amylin (SEQ ID NO: 148) (125 nmol/kg),either alone or in combination over four weeks.

FIG. 12 depicts the effects of the indicated engineered polypeptidesdescribed herein on blood glucose upon administration to STZ-inducedT1DM mice as described in Example 16.

FIG. 13 depicts the effects of the indicated engineered polypeptidesdescribed herein on Hemoglobin A1C upon administration to STZ-inducedT1DM mice as described in Example 16.

FIG. 14 depicts the effects of the indicated engineered polypeptidesdescribed herein on food intake and body weight upon administration toSTZ-induced T1DM mice as described in Example 16.

FIG. 15 depicts the effects of the indicated engineered polypeptidesdescribed herein, with and without a low dose of insulin, on bloodglucose upon administration to STZ-induced T1DM mice as described inExample 16.

FIG. 16 depicts the effects of the indicated engineered polypeptidesdescribed herein, with and without a low dose of insulin, on HemoglobinA1C upon administration to STZ-induced T1DM mice as described in Example16.

FIG. 17 depicts the effects of the indicated engineered polypeptidesdescribed herein, with and without a low dose of insulin, on food intake(% vehicle-corrected) and change in body weight (% vehicle-corrected)upon administration to STZ-induced T1DM mice as described in Example 16.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Obesity” and “overweight” refer to mammals having a weight greater thannormally expected, and may be determined by, e.g., physical appearance,body mass index (BMI) as known in the art, waist-to-hip circumferenceratios, skinfold thickness, waist circumference, and the like. TheCenters for Disease Control and Prevention (CDC) define overweight as anadult human having a BMI of 25 to 29.9; and define obese as an adulthuman having a BMI of 30 or higher. Additional metrics for thedetermination of obesity exist. For example, the CDC states that aperson with a waist-to-hip ratio greater than 1.0 is overweight.

“Lean body mass” refers to the fat-free mass of the body, i.e., totalbody weight minus body fat weight is lean body mass. Lean body mass canbe measured by methods such as hydrostatic weighing, computerizedchambers, dual-energy X-ray absorptiometry, skin calipers, magneticresonance imaging (MRI) and bioelectric impedance analysis (BIA) asknown in the art.

“Mammal” refers to warm-blooded animals that generally have fur or hair,that give live birth to their progeny, and that feed their progeny withmilk. Mammals include humans; companion animals (e.g., dogs, cats); farmanimals (e.g., cows, horses, sheep, pigs, goats); wild animals; and thelike. In one embodiment, the mammal is a female. In one embodiment, themammal is a female human. In one embodiment, the mammal is a cat or dog.In one embodiment, the mammal is a diabetic mammal, e.g., a human havingtype 2 diabetes. In one embodiment, the mammal is an obese diabeticmammal, e.g., an obese mammal having type 2 diabetes. The term “subject”in the context of methods described herein refers to a mammal.

“Fragment” in the context of polypeptides refers herein in the customarychemical sense to a portion of a polypeptide. For example, a fragmentcan result from N-terminal deletion or C-terminal deletion of one ormore residues of a parent polypeptide, and/or a fragment can result frominternal deletion of one or more residues of a parent polypeptide.“Fragment” in the context of an antibody refers to a portion of anantibody which can be linked to a biologically active molecule tomodulate solubility, distribution within a subject, and the like. Forexample, leptin A200 described herein is a conjugate of an Fc antibodyfragment with a leptin, as known in the art. See e.g. WO 98/28427 andUS2007/002084. The term “parent” in the context of polypeptides refers,in the customary sense, to a polypeptide which serves as a referencestructure prior to modification, e.g., insertion, deletion and/orsubstitution. The term “conjugate” in the context of engineeredpolypeptides described herein refers to covalent linkage betweencomponent polypeptides, e.g., ABD, HD1 and the like. The term “fusion”in the context of engineered polypeptides described herein refers tocovalent linkage between component polypeptides, e.g., ABD, HD1 and thelike, via either or both terminal amino or carboxy functional group ofthe peptide backbone. Engineered polypeptides can be synthetically orrecombinantly made. Typically, fusions are made using recombinantbiotechnology, however, can also be made by chemical synthesis andconjugation methods known in the art.

“Analog” as used herein in the context of polypeptides refers to acompound that has insertions, deletions and/or substitutions of aminoacids relative to a parent compound. An analog may have superiorstability, solubility, efficacy, half-life, and the like. In someembodiments, an analog is a compound having at least 50%, for example50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even higher,sequence identity to the parent compound.

“Identity,” “sequence identity” and the like in the context of comparingtwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 50% identity, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window or designated region) asmeasured using a sequence comparison algorithms as known in the art, forexample BLAST or BLAST 2.0. This definition includes sequences that havedeletions and/or additions, as well as those that have substitutions, aswell as naturally occurring, e.g., polymorphic or allelic variants, andman-made variants. In preferred algorithms, account is made for gaps andthe like, as known in the art. For sequence comparison, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated if necessary, and sequence algorithm program parameters aredesignated. Preferably, default program parameters can be used, oralternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the testsequences relative to the reference sequence, based on the programparameters. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection. See e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 1995 supplement)). Preferredexamples of algorithms that are suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al., 1977, Nuci.Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol.215:403-410. BLAST and BLAST 2.0 are used, as known in the art, todetermine percent sequence identity for the nucleic acids and proteinsof the invention. Software for performing BLAST analyses is publiclyavailable through the web site of the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., Id.). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, e.g., for nucleotide sequences,the parameters M (reward score for a pair of matching residues;always>0) and N (penalty score for mismatching residues; always<0). Foramino acid sequences, a scoring matrix is used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The term “about” in the context of a numeric value refers to +/−10% ofthe numeric value, unless expressly indicated otherwise.

The terms “peptide” and “polypeptide” in the context of components ofthe engineered polypeptides described herein are synonymous.

II. Compounds

In a first aspect, engineered polypeptide compounds are provided whichinclude an albumin binding domain (ABD) polypeptide and at least onepolypeptide hormone domain (HD1). The terms “albumin binding domain,”“ABD” and the like refer to polypeptides capable of binding albumin asdescribed herein. The terms “hormone domain,” “hormone domainpolypeptide” and the like refer to a polypeptide capable of eliciting abiological response in a subject. Exemplary hormone domains include, butare not limited to, a leptin, an analog of a leptin or an activefragment thereof, but could be a leptin derivative such as a PEGylatedderivative.

It was surprisingly found that a leptin, a leptin analog, a activeleptin fragment, or a leptin derivative thereof can be fused to anvery-high-affinity albumin binding domain (ABD) derived from thealbumin-binding domains of bacterial proteins as described herein, whileretaining sufficient leptin biological activity and having an extendedduration of action, for example of at least 3 days and even 5 days in arodent, which translates to at least a one week duration or longer in ahuman subject. This was surprising in part because such ABD peptideshave not been extensively demonstrated to be a robust platform as atherapeutic protein carrier, they are relatively hydrophobic which couldinteract adversely with an attached therapeutic peptide, and were notable to act as a carrier for at least one family of peptide hormones.For instance, rat amylin compounds (e.g., SEQ ID NO:108), whenconjugated or fused to the ABDs described herein, did not display anysignificant or long-acting in vivo activity in the same rodent models inwhich various leptin engineered polypeptide constructs of the inventionwere found to be active and with long duration of action.

Furthermore, the therapeutic conjugate or fusion compounds hereinsurprisingly have retained albumin binding affinity and specificitywhile having lower immunogenicity and leptin therapeutic activity. Thecompounds are surprisingly active despite the absence of aplasma-protease cleavage site between the leptin, leptin analog, activeleptin fragment, or leptin derivative and the ABD. Further surprising,the therapeutic compounds are believed active even when bound toalbumin. The ABD compounds described herein provide albumin bindingaffinity and specificity while having lower immunogenicity thanpreviously described ABD compounds, which were based on the albuminbinding region of Streptococcal protein G strain 148 (G148) and inJonsson et al. (Protein Eng. Design & Selection, 2008, 21:515-527).Recently, a few T- and B-cell epitopes were experimentally identifiedwithin the albumin binding region of Streptococcal protein G strain 148(G148) (Goetsch et al, Clin. Diagn. Lab. Immunol. 10:125-32, 2003). Theauthors were interested in utilizing the T-cell epitopes of G148 invaccines, i.e. to utilize the inherent immune-stimulatory property ofthe albumin binding region. Goetsch et al. additionally found a B-cellepitope, i.e. a region bound by antibodies after immunization, in thesequence of G148. In pharmaceutical compositions for humanadministration no immune-response is desired. Therefore, the albuminbinding domain G148 is as such not preferred for use in suchcompositions due to its abovementioned immune-stimulatory properties.

Biologically Active Components.

Biologically active compound components contemplated for use in thecompounds and methods described herein include leptins. The terms“biologically active compound” and the like refer in the customary senseto compounds, e.g., polypeptides and the like, which can elicit abiological response.

Leptins.

“Leptins” and “a leptin” means: leptins, leptin active fragments, leptinanalogs, and leptin derivatives; and a leptin, a leptin active fragment,a leptin analog, and a leptin derivative; respectively. Accordingly,unless otherwise noted, reference to “leptins” is meant to encompassleptins, leptin active fragments, leptin analogs, and leptinderivatives, as disclosed herein. Similarly, unless otherwise noted,reference to “a leptin” is meant to encompass a leptin, a leptin activefragment, a leptin analog, and a leptin derivative, as disclosed herein.Exemplary such leptins which may be employed in the design, preparation,and use of the engineered polypeptides disclosed herein include thosewhich elicit one or more biological responses known in the art to beelicited when leptins are administered to subjects (see, e.g., publishedU.S. Patent application Nos. US 2007/0020284 and US 2008/0207512, U.S.Pat. Nos. 6,309,853, and 7,183,254, and PCT Published Application Nos.WO 96/005309, WO 98/28427, and WO 2009/064298), such as: reduction offood intake, reduction of body weight, reduction of body weight gain,induction of satiety, reduction of caloric availability, reduction ofcaloric efficiency, reduction of metabolic plateau, increase in insulinsensitivity, reduction of hyperlipidemia, correction of dyslipidemia,reduction of hypertriglyceridemia, amelioration of obesity, ameliorationof overweight, amelioration of diabetes mellitus (including type Idiabetes, type II diabetes, and gestational diabetes), amelioration ofinsulin resistance, amelioration of lipodystrophy conditions associatedtherewith, as well as other biological responses known in the art to beelicited upon administration of a leptin (see, e.g., published U.S.Patent Application Nos. US 2007/0020284 and US 2008/0207512, U.S. Pat.Nos. 6,309,853, and 7,183,254, and PCT Published Application Nos. WO96/005309, WO 98/28427, and WO 2009/064298.

Exemplary leptins suitable for the design, preparation, and use of theengineered polypeptides described herein include, but are not limitedto, the compounds described in U.S. Pat. Nos. 5,594,101, 5,851,995,5,691,309, 5,580,954, 5,554,727, 5,552,523, 5,559,208, 5,756,461,6,309,853, published U.S. Patent application No. US 2007/0020284, andPCT Published Application Nos. WO 96/23517, WO 96/005309, WO 98/28427,WO 2004/039832, WO 98/55139, WO 98/12224, and WO 97/02004, each of whichis incorporated herein in its entirety and for all purposes. Methods toassay for leptin activities and biological responses in vitro and invivo, including satiety, food intake inhibition activity and weight lossactivity, are known in the art and are described herein and also in theabove references and other references recited herein.

Any leptin, leptin analog, leptin active fragment, or leptin derivativeknown in the art may be employed in order to prepare and use engineeredpolypeptides as disclosed herein throughout. Representative leptins,leptin analogs, leptin active fragments, and leptin derivativescontemplated for use in the engineered polypeptides and methodsdescribed herein also include the following:

Mature Murine Leptins:

(SEQ ID NO: 1) VPIQKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSP GC, wherein Xaa atposition 28 is Q or absent.

Mature Murine Leptin Form 1:

(SEQ ID NO: 143) VPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Murine Leptin Form 2:

(SEQ ID NO: 144) VPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Murine Leptins with N-Terminal Methionine:

(SEQ ID NO: 2) MVPIQKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSP GC, wherein Xaa atposition 29 is Q or absent.

Mature Murine Leptin Form 1 with N-Terminal Methionine:

(SEQ ID NO: 145) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Murine Leptin Form 2 with N-Terminal Methionine:

(SEQ ID NO: 146) MVPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Porcine Leptin:

(SEQ ID NO: 3) VPIWRVQDDTKTLIKTIVTRISDISHMQSVSSKQRVTGLDFIPGLHPVLSLSKMDQTLAIYQQILTSLPSRNVIQISNDLENLRDLLHLLASSKSCPLPQARALETLESLGGVLEASLYSTEVVALSRLQGALQDMLRQLDLSPGC.

Mature Porcine Leptin with N-Terminal Methionine:

(SEQ ID NO: 4) MVPIWRVQDDTKTLIKTIVTRISDISHMQSVSSKQRVTGLDFIPGLHPVLSLSKMDQTLAIYQQILTSLPSRNVIQISNDLENLRDLLHLLASSKSCPLPQARALETLESLGGVLEASLYSTEVVALSRLQGALQDMLRQLDLSPGC.

Mature Bovine Leptins:

(SEQ ID NO: 5) VPICKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKMDQTLAIYQQILTSLPSRNVVQISNDLENLRDLLHLLAASKSCPLPQVRALESLESLGVVLEASLYSTEVVALSRLQGSLQDMLRQLDLSPG C, wherein Xaa atposition 28 is Q or absent.

Mature Bovine Leptins with N-Terminal Methionine:

(SEQ ID NO: 6) MVPICKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKMDQTLAIYQQILTSLPSRNVVQISNDLENLRDLLHLLAASKSCPLPQVRALESLESLGVVLEASLYSTEVVALSRLQGSLQDMLRQLDLSPG C, wherein Xaa atposition 29 is Q or absent.

Unprocessed Full-Length Human Leptin (i.e., Includes 21-ResidueN-Terminal Signal Sequence):

(SEQ ID NO: 7) MHWGTLCGFLWLWPYLFYVQAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRL QGSLQDMLWQLDLSPGC

Mature Human Leptins (with N-Terminal 21 Amino Acid Signal SequenceRemoved):

(SEQ ID NO: 8) VPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLS PGC, wherein: Xaa atposition 27 is T or A; and Xaa at position 28 is Q or absent.

Mature Human Leptins with N-Terminal Methionine:

(SEQ ID NO: 9) MVPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDL SPGC, wherein: Xaa atposition 28 is T or A; and Xaa at position 29 is Q or absent.

Mature Rhesus Leptin:

(SEQ ID NO: 10) VPIQKVQSDTKTLIKTIVTRINDISHTQSVSSKQRVTGLDFIPGLHPVLTLSQMDQTLAIYQQILINLPSRNVIQISNDLENLRDLLHLLAFSKSCHLPLASGLETLESLGDVLEASLYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Rhesus Leptin with N-Terminal Methionine:

(SEQ ID NO: 11) MVPIQKVQSDTKTLIKTIVTRINDISHTQSVSSKQRVTGLDFIPGLHPVLTLSQMDQTLAIYQQILINLPSRNVIQISNDLENLRDLLHLLAFSKSCHLPLASGLETLESLGDVLEASLYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Rat Leptin:

(SEQ ID NO: 12) VPIHKVQDDTKTLIKTIVTRINDISHTQSVSARQRVTGLDFIPGLHPILSLSKMDQTLAVYQQILTSLPSQNVLQIAHDLENLRDLLHLLAFSKSCSLPQTRGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDLSPEC.

Mature Rat Leptin with N-Terminal Methionine:

(SEQ ID NO: 13) MVPIHKVQDDTKTLIKTIVTRINDISHTQSVSARQRVTGLDFIPGLHPILSLSKMDQTLAVYQQILTSLPSQNVLQIAHDLENLRDLLHLLAFSKSCSLPQTRGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDLSPEC.

Mature Platypus Leptin:

The Mature Platypus Leptin Sequence Follows:

(SEQ ID NO: 14) ISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNIEKQLDHIQG.

Unprocessed Full-Length Platypus Leptin (i.e., Includes 21-ResidueN-Terminal Signal Sequence):

A full length sequence of platypus leptin, including a 21-residueN-terminal signal sequence follows:

(SEQ ID NO: 15) MRCILLYGFLCVWQHLYYSHPISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRL RKSLKNIEKQLDHIQG.

Mature Human Leptin Form 1:

(SEQ ID NO: 16) VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 2:

(SEQ ID NO: 17) VPIQKVQDDTKTLIKTIVTRINDISHAQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 3:

(SEQ ID NO: 18) VPIQKVQDDTKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 4:

(SEQ ID NO: 19) VPIQKVQDDTKTLIKTIVTRINDISHASVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 1 with N-Terminal Methionine (Also Known asMetreleptin, or A100):

(SEQ ID NO: 20) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 2 with N-Terminal Methionine:

(SEQ ID NO: 21) MVPIQKVQDDTKTLIKTIVTRINDISHAQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 3 with N-Terminal Methionine:

(SEQ ID NO: 22) MVPIQKVQDDTKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 4 with N-Terminal Methionine:

(SEQ ID NO: 23) MVPIQKVQDDTKTLIKTIVTRINDISHASVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Seal Leptin:

(SEQ ID NO: 24) PIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with Amino Acids 71-92 Replaced with Amino Acids 73-94(Helix 3) of Metreleptin, Respectively:

(SEQ ID NO: 25) PIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with Amino Acids 30 and 71-92 Replaced with Amino Acids 32and 73-94 (Helix 3) of Metreleptin, Respectively:

(SEQ ID NO: 26) PIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with N-Terminal Methionine:

(SEQ ID NO: 27) MPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with N-Terminal Methionine, and with Amino Acids 71-92Replaced with Amino Acids 73-94 (Helix 3) of Metreleptin, Respectively:

(SEQ ID NO: 28) MPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with N-Terminal Methionine, and with Amino Acids 30 and71-92 Replaced with Amino Acids 32 and 73-94 (Helix 3) of Metreleptin,Respectively:

(SEQ ID NO: 29) MPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with Amino Acids 71-92 Replaced with Amino Acids 73-94(Helix 3) of Metreleptin, and with Amino Acids 23-49 Replaced with AminoAcids 25-51 (AB Loop) of Metreleptin, Respectively:

(SEQ ID NO: 680) PIQRVQDDTKTLIKTIITRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC

Leptin A200:

Leptin A200 is an Fc antibody fragment condensation product with leptin,as known in the art. See e.g., Lo et al., 2005, Protein Eng. Design &Selection, 18:1-10. The amino acid sequence of A200 is as follows:

(SEQ ID NO: 30) MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC

Leptin A300:

Leptin A300 is metreleptin with substitutions W101Q and W139Q(N-terminal ¹Met counted as residue 1):

(SEQ ID NO: 31) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC.

Leptin A400:

Leptin A400 is metreleptin with the serine residue at position 78replaced with a cysteine residue, as set forth following:

(SEQ ID NO: 32) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQICNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC;to which a 20 kilodalton (kDa) PEG moiety has been attached via thecysteine residue at position 78.

Leptin A500:

Research by a number of investigators including the inventors hasfocused on the effects on aggregation of residue substitution in leptin.See e.g., Ricci et al., 2006. “Mutational approach to improve physicalstability of protein therapeutics susceptible to aggregation: Role ofaltered conformation in irreversible precipitation,” Book Chapter. In:MISBEHAVING PROTEINS: PROTEIN (MIS)FOLDING, AGGREGATION, AND STABILITY,Murphy R M, Tsai A M, Eds., New York. Springer. pp. 331-350, which isincorporated herein by reference and for all purposes. Accordingly,leptin A500 with sequence following has been used in certain compoundsand methods described herein:

(SEQ ID NO: 33) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC.

Leptin A100 Variants:

Variants of Leptin A100 with the following residue substitutions follow:

D41E, H98S, W101Q, D109E, G113E, M137I, W139Q and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 664). H98S,W101Q, A102T, G113E, M137I, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 665). H98S,W101Q, G113E, M137I, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 666).W101Q, G113E, M137I, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 667). H98S,W101Q, M137I, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 668). W101Q,G113E, M137I, W139Q, L143V, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 669). H98S,W101Q, A102T, M137I, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQTSGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 670). H98S,W101Q, D109E, G13E, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPEC (SEQ ID NO: 671). W101Q,M137I, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC (SEQ ID NO: 672). W101Q,M137I, W139Q, L143V, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 673). H98S,W101Q, A102T, M137I, W139Q, L143V, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQTSGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDVSPEC (SEQ ID NO: 674). H98S,W101Q, A102T, G113E, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQTSGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPEC (SEQ ID NO: 675).W101Q, G113E, and W139Q:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 676).W101Q, G113E, W139Q, and G146E:MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPEC (SEQ ID NO: 677).

Any of the above leptins, leptin analogs or their active fragments, aswell as leptins as described below, are suitable for use in the presentengineered polypeptides, with or without a linker to the ABD.

Albumin Binding Domain (ABD) Peptides.

For previously disclosed albumin binding domains derived fromStreptococcal protein G strain 148 (G148) and for some variants having ahigh affinity to albumin, e.g. WO09/016043, the higher affinity wasachieved at the cost of reduced thermal stability. In addition, it hasbeen reported that T- and B-cell epitopes were experimentally identifiedwithin the albumin binding region of G148 (Goetsch et al, Clin Diagn LabImmunol 10:125-32, 2003). The authors behind the study were interestedin utilizing the T-cell epitopes of G148 in vaccines, i.e. to utilizethe inherent immune-stimulatory property of the albumin binding region.Goetsch et al. additionally found a B-cell epitope, i.e. a region boundby antibodies after immunization, in the sequence of G148. Therefore,the albumin binding domain G148 and polypeptides derived from G148, andthus fusion/conjugates containing them, risk the abovementionedimmune-stimulatory properties. In pharmaceutical compositions for humanadministration no (or reduced) immune-response is desired.

The above drawbacks and deficiencies of such fusions and/or conjugatesare overcome or reduced by the use of the albumin binding domain (ABD)peptides disclosed herein for use in the engineered polypeptides of theinvention. Accordingly, in one embodiment the albumin binding domainpolypeptide comprising the long-duration engineered polypeptideconjugate or fusion described herein is a three-helix bundle proteindomain, which comprises an albumin binding motif and additionalsequences comprising the three-helix configuration. Such ABDs are thosewith comparably high affinity for albumin and derive fromalbumin-binding domains of bacterial protein G of Streptococcus strainG148, yet are modified as described herein to further provide desirableimmunological properties, e.g. reduced immunogenicity. Such ABDs aredescribed in PCT Published Appl. No. WO 2012/004384, incorporated hereinby reference in its entirety and for all purposes. As such, ABD peptidescontemplated for the engineered polypeptides described herein aresuperior to those having the albumin binding sequence as described byJonsson et al. (Protein Eng. Design & Selection, 2008, 21:515-527), andthose ABD peptides further described in PCT Published Appl. No.WO2009/016043. To the albumin binding domain polypeptide describedherein is fused a leptin or analog or active fragment thereof to createthe engineered polypeptide described herein. An albumin binding domainpolypeptide suitable for conjugation or fusion to a leptin compound cancomprise the ABD amino acid sequence which comprises the sequenceselected from:

formula (i) (SEQ ID NO: 300) LA X3 AK X6 X7 AN X10 ELD X14 YGVSDF YKRLIX26 KAKT VEGVEALK X39 X40 IL X43 X44 LPwherein independently of each otherX3 is selected from E, S, Q and C;X6 is selected from E, S and C;X7 is selected from A and S;X10 is selected from A, S and R;X14 is selected from A, S, C and K;X26 is selected from D and E;X39 is selected from D and E;X40 is selected from A and E;X43 is selected from A and K;X44 is selected from A, S and E;the leucine at position 45 is present or absent; andthe proline at position 46 is present or absent; and

formula (ii) an amino acid sequence which has at least 95% identity tothe sequence defined in (i),

with the proviso that X₇ is not L, E or D;

or alternatively,

with the proviso that the amino acid sequence is not defined by thefollowing sequence, as defined in PCT Published Application No. WO2009/016043: LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY GVSD X₅ YK X₈X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ ILAALP (SEQ ID NO: 679)

wherein

independently of each other,

X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K;

X_(e) is selected from D and K; and

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and

X₂₅ is selected from H, E and D.

In a further embodiment of the albumin binding polypeptide according tothe first aspect above—the formula (i) or (ii), X6 is E. In anotherembodiment of the albumin binding polypeptide according to this aspect,X3 is S. In another embodiment of the albumin binding polypeptideaccording to this aspect, X3 is E. In another embodiment of the albuminbinding polypeptide according to this aspect, X7 is A. In anotherembodiment of the albumin binding polypeptide according to this aspect,X14 is S. In another embodiment of the albumin binding polypeptideaccording to this aspect, X14 is C. In another embodiment of the albuminbinding polypeptide according to this aspect, X10 is A. In anotherembodiment of the albumin binding polypeptide according to this aspect,X10 is S. In another embodiment of the albumin binding polypeptideaccording to this aspect, X10 is R. In another embodiment of the albuminbinding polypeptide according to this aspect, X26 is D. In anotherembodiment of the albumin binding polypeptide according to this aspectX39 is D. In another embodiment of the albumin binding polypeptideaccording to this aspect X39 is E. In another embodiment of the albuminbinding polypeptide according to this aspect X40 is A. In anotherembodiment of the albumin binding polypeptide according to this aspectX43 is A. In another embodiment of the albumin binding polypeptideaccording to this aspect X44 is A. In another embodiment of the albuminbinding polypeptide according to this aspect X44 is S. In anotherembodiment of the albumin binding polypeptide according to this aspectthe leucine at position 45 is present or absent. In another embodimentof the albumin binding polypeptide according to this aspect proline atposition 46 is absent.

In a further preferred embodiment albumin binding domain polypeptidesuitable for conjugation or fusion to a leptin compound can comprise theABD amino acid sequence selected from:

formula (iii) (SEQ ID NO: 678) LA X3 AK X6 X7 AN X10 ELD X14 YGVSDFYKRLIDKAKT VEGVEALKDA ILAALPwherein independently of each otherX3 is selected from E, S, Q and C;X6 is selected from E, S and C;X7 is selected from A and S;X10 is selected from A, S and R;X14 is selected from A, S, C and K;the leucine at position 45 is present or absent; andthe proline at position 46 is present or absent; andformula (iv) an amino acid sequence which has at least 95% identity tothe sequence defined in (iii),

with the proviso that X₇ is not L, E or D;

or alternatively,

with the proviso that the amino acid sequence is not defined by thefollowing sequence, as defined in PCT Published Application No. WO2009/016043: LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY GVSD X₅ YK X₈X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ ILAALP (SEQ ID NO: 679)

wherein

independently of each other,

X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K;

X_(e) is selected from D and K; and

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and

X₂₅ is selected from H, E and D.

In a further embodiment of the albumin binding polypeptide according tothis aspect—formula (iii) or (iv), X6 is E. In another embodiment of thealbumin binding polypeptide according to this aspect, X3 is S. Inanother embodiment of the albumin binding polypeptide according to thisaspect, X3 is E. In another embodiment of the albumin bindingpolypeptide according to this aspect, X7 is A. In another embodiment ofthe albumin binding polypeptide according to this aspect, X14 is S. Inanother embodiment of the albumin binding polypeptide according to thisaspect, X14 is C. In another embodiment of the albumin bindingpolypeptide according to this aspect, X10 is A. In another embodiment ofthe albumin binding polypeptide according to this aspect, X10 is S. Inanother embodiment of the albumin binding polypeptide according to thisaspect, X10 is R. In another embodiment of the albumin bindingpolypeptide according to this aspect the leucine at position 45 ispresent or absent. In a further embodiment the C-terminal proline ispresent. In a further embodiment the proline at is absent.

In a further embodiment of any one of the formulas (i) to (iv) the ABDcomprises a one or more N-terminal helix-capping amino acids, and in afurther embodiment the helix-capping amino acid may be serine, or may beglycine-serine. Accordingly for each albumin binding domain sequencedisclosed herein, including those in the figures and sequenced listing,also specifically contemplated for all aspects as disclosed herein inthe engineered polypeptide, are albumin binding domains corresponding tothe ABD of any one of the formulas (i) to (iv) contained therein, theirSer-ABD, Gly-Ser-ABD, Gly-ABD, Ala-ABD and their des-C-terminal-prolinesequences.

Thus, modified variants of (i) or (iii), which are such that theresulting sequence is at least 95% identical to a sequence belonging tothe class defined by (i) or (iii), are also encompassed. For example, itis possible that an amino acid residue belonging to a certain functionalgrouping of amino acid residues (e.g. hydrophobic, hydrophilic, polaretc) could be exchanged for another amino acid residue from the samefunctional group.

The above defined class of sequence related ABD polypeptides having abinding affinity for albumin is derived from a common parent polypeptidesequence, which folds into a three alpha helix bundle domain. Morespecifically, the polypeptides as described above are derived from amodel building based on a structure of a complex between serum albuminand the albumin binding domain G148-GA3 (Lejon et al, J. Biol. Chem.279:42924-8, 2004), as well as analyses of binding and structuralproperties of a number of mutational variants of the common parentpolypeptide sequence. The above defined amino acid sequence of any oneof formulas (i) to (iv) comprises amino acid substitutions, as comparedto the parent polypeptide sequence, that result in a class ofpolypeptides which are expected to fold into an almost identical threehelix bundle domain. While the parent polypeptide sequence alreadycomprises a binding surface for interaction with albumin, that bindingsurface is modified by some of the substitutions according to the abovedefinition. The substitutions according to the above definition providean improved albumin binding ability as compared to the parentpolypeptide sequence. Importantly and surprisingly, the substitutionsaccording to the above definition provide enhanced immunologicalproperties, in addition to retaining and/or improving strong affinityfor albumin.

Accordingly, the albumin binding polypeptides according to the firstaspect of the disclosure exhibit a set of characteristics, which, forexample, make them suitable for use as fusion or conjugate partners fortherapeutic molecules for human administration. Importantly andsurprisingly, the albumin binding polypeptides according to the presentdisclosure demonstrate, for example in comparison with related albuminbinding polypeptides such as the albumin binding domain G148-GA3 and thealbumin binding polypeptides disclosed in WO09/016043, at least five ofthe following six characteristics:

(1) The polypeptides display a different surface compared to, forexample, G148-GA3 and other bacterially derived albumin binding domains.The difference may decrease or eliminate any risk for antibody reactionsin a subject, such as a human, which has been previously exposed to suchbacterial proteins.(2) The polypeptides comprise fewer potential T-epitopes than, forexample, G148-GA3 and other related, but different, mutational variantsof the common parent polypeptide sequence, and hence exhibit low and/orlower immunogenicity when administered to a subject, such as a human.(3) The polypeptides display lower reactivity with circulatingantibodies when administered to a subject, such as a human. Thus, byamino acid substitutions in the surface of the polypeptides exposed tocirculating antibodies, i.e. in the polypeptide surface not involved inthe binding interaction with albumin, antibody cross-reactivity isreduced as compared to, for example, antibody cross-reactivity caused byG148-GA3 as measured in a test set of human sera.(4) The polypeptides have a high albumin binding ability, both in termsof a higher binding affinity, as defined by a K_(D) value, and in termsof a slower off-rate, as defined by a koff value, than, for example,known naturally occurring albumin binding polypeptides, such as thealbumin binding domains derived from bacterial proteins.(5) The polypeptides comprise fewer amino acid residues that areassociated with stability problems of polypeptides than, for example,known naturally occurring albumin binding polypeptides, such as thealbumin binding domains derived from bacterial proteins. Thus, thepolypeptides comprise, for example, no oxidation-prone methionines ortryptophans and only one asparagine.(6) The polypeptides have a higher structural stability, as defined by amelting point of above 55° C., than previous albumin bindingpolypeptides, such as those disclosed in WO09/016043.

In one embodiment, the albumin binding polypeptide of theconjugate/fusions according to the first aspect display all six of theabove listed characteristics. In another embodiment, the albumin bindingpolypeptide according to the first aspect displays, when bound toalbumin, a more hydrophilic profile than, for example, previous albuminbinding polypeptides, such as those disclosed in WO09/016043. Thesurface of the albumin binding polypeptide which is exposed to thesurroundings when the polypeptide interacts with albumin comprises feweramino acid residues that confer surface hydrophobicity.

The ABD peptide binds to albumin with a K_(D) value of the interactionthat is at most 1×10⁻⁶ M and even more preferably at most 1×10⁻⁹ M (eventighter affinity). More preferably the K_(D) value of the interactionthat is at most 1×10⁻¹⁰ M, even more preferably is at most 1×10⁻¹¹ M,yet even more preferably is at most 1×10⁻¹² M, and even further is atmost 1×10⁻¹³ M. The values are most preferably for affinity to humanserum albumin (“HSA”).

In embodiments of the present invention wherein the motif “forms part ofa three-helix bundle protein domain,” this is understood to mean thatthe sequence of the albumin binding motif is “inserted” into or“grafted” onto or “fused” to the sequence of the naturally occurring (orotherwise original) three-helix bundle domain, such that the motifreplaces a similar structural motif in the original domain. For exampleand without wishing to be bound by theory, the motif is thought toconstitute two of the three helices of a three-helix bundle, and canreplace such a two-helix motif within any three-helix bundle. Thereplacement of two helices of the three-helix bundle domain by the twomotif helices disclosed herein is performed so as not to affect thebasic structure of the polypeptide. That is, the overall folding of thebackbone of the polypeptide according to this embodiment of theinvention will be substantially the same as that of the three-helixbundle protein domain of which it forms a part, e.g. having the sameelements of secondary structure in the same order etc. Thus, a motifuseful to the engineered polypeptides herein can form part of athree-helix bundle domain if the polypeptide according to thisembodiment has the same fold as the original domain, implying that thebasic structural properties are shared, those properties e.g. resultingin similar CD spectra.

The terms “albumin binding” and “binding affinity for albumin” as usedin this specification refer to a property of a polypeptide which may betested for example by the use of surface plasmon resonance technology,such as in a Biacore instrument. For example as described in theexamples below, albumin binding affinity may be tested in an experimentin which albumin, or a fragment thereof, is immobilized on a sensor chipof the instrument, and the sample containing the polypeptide to betested is passed over the chip. Alternatively, the polypeptide to betested is immobilized on a sensor chip of the instrument, and a samplecontaining albumin, or a fragment thereof, is passed over the chip.Albumin may, in this regard, be a serum albumin from a mammal, such ashuman serum albumin. The skilled person may then interpret the resultsobtained by such experiments to establish at least a qualitative measureof the binding affinity of the polypeptide for albumin. If aquantitative measure is desired, for example to determine a K_(D) valuefor the interaction, surface plasmon resonance methods may also be used.Binding values may for example be defined in a Biacore2000 instrument(GE Healthcare). Albumin is suitably immobilized on a sensor chip of themeasurement, and samples of the polypeptide whose affinity is to bedetermined are prepared by serial dilution and injected. K_(D) valuesmay then be calculated from the results using for example the 1:1Langmuir binding model of the BIAevaluation 4.1 software provided by theinstrument manufacturer (GE Healthcare).

In one embodiment, the albumin binding polypeptide according to thisaspect binds to albumin such that the k_(off) value of the interactionis at most 5×10⁻⁵ s⁼¹, such as at most 5×10⁻⁶ s⁻¹.

In another embodiment, the amino acid sequence of the albumin bindingpolypeptide is selected from any one of SEQ ID NO:301-452, SEQ IDNO:455-463, and SEQ ID NO:500-593. More specifically, the amino acidsequence is selected from SEQ ID NO:304-305, SEQ ID NO:307-308, SEQ IDNO:310-311, SEQ ID NO:313-314, SEQ ID NO:316-317, SEQ ID NO:319-320, SEQID NO:322-323, SEQ ID NO:325-326, SEQ ID NO:328-329, SEQ ID NO:331-332,SEQ ID NO:334-335, SEQ ID NO:337-338, SEQ ID NO:341-342 and SEQ IDNO:349-350.

In one embodiment, the albumin binding polypeptide according to thisaspect further comprises one or more additional amino acid residuespositioned at the N- and/or the C-terminal of the sequence defined in(i) or (iii). These additional amino acid residues may play a role inenhancing the binding of albumin by the polypeptide, and improving theconformational stability of the folded albumin binding domain, but mayequally well serve other purposes, related for example to one or more ofproduction, purification, stabilization in vivo or in vitro, coupling,labeling or detection of the polypeptide, as well as any combinationthereof. Such additional amino acid residues may comprise one or moreamino acid residue(s) added for purposes of chemical coupling, e.g. toan HD1.

The amino acids directly preceding or following the alpha helix at theN- or C-terminus of the amino acid sequence (i) or (iii) may thus in oneembodiment affect the conformational stability. One example of an aminoacid residue which may contribute to improved conformational stabilityis a serine residue positioned at the N-terminal of the amino acidsequence (i) or (iii) as defined above. The N-terminal serine residuemay in some cases form a canonical S—X—X-E capping box, by involvinghydrogen bonding between the gamma oxygen of the serine side chain andthe polypeptide backbone NH of the glutamic acid residue. ThisN-terminal capping may contribute to stabilization of the first alphahelix of the three helix domain constituting the albumin bindingpolypeptide according to the first aspect of the disclosure.

Thus, in one embodiment, the additional amino acids comprise at leastone serine residue at the N-terminal of the polypeptide. The amino acidsequence is in other words preceded by one or more serine residue(s). Inanother embodiment of the albumin binding polypeptide, the additionalamino acids comprise a glycine residue at the N-terminal of thepolypeptide. It is understood that the amino acid sequence (i) or (iii)may be preceded by one, two, three, four or any suitable number of aminoacid residues. Thus, the amino acid sequence may be preceded by a singleserine residue, a single glycine residue or a combination of the two,such as a glycine-serine (GS) combination or a glycine-serine-serine(GSS) combination. Examples of albumin binding polypeptides comprisingadditional amino residues at the N-terminal are set out in SEQ IDNO:445-463, such as in SEQ ID NO:445-448 and SEQ ID NO:462-463. In yetanother embodiment, the additional amino acid residues comprise aglutamic acid at the N-terminal of the polypeptide as defined by thesequence formula (i) or (iii).

Similarly, C-terminal capping may be exploited to improve stability ofthe third alpha helix of the three helix domain constituting the albuminbinding polypeptide. The C-terminal proline residue present at theC-terminal of the amino acid sequence defined in (i) or (iii) may atleast partly function as a capping residue. A lysine residue followingthe proline residue at the C-terminal may contribute to furtherstabilization of the third helix of the albumin binding polypeptide, byhydrogen bonding between the epsilon amino group of the lysine residueand the carbonyl groups of the amino acids located two and threeresidues before the lysine in the polypeptide backbone, e.g. thecarbonyl groups of the leucine and alanine residues of the amino acidsequence defined in (i) or (iii). Thus, in one embodiment, theadditional amino acids comprise a lysine residue at the C-terminal ofthe polypeptide.

As discussed above, the additional amino acids may be related to theproduction of the albumin binding polypeptide. In particular, one ormore optional amino acid residues following the C-terminal proline mayprovide advantages when the albumin binding polypeptide according to thefirst aspect is produced by chemical peptide synthesis. Such additionalamino acid residues may for example prevent formation of undesiredsubstances, such as diketopiperazine at the dipeptide stage of thesynthesis. One example of such an amino acid residue is glycine. Thus,in one embodiment, the additional amino acids comprise a glycine residueat the C-terminal of the polypeptide, directly following the prolineresidue or following an additional lysine and/or glycine residue asaccounted for above.

Examples of albumin binding polypeptides comprising additional aminoacid residues at the C-terminal are set out in SEQ ID NO:445-452, suchas in SEQ ID NO:449-450. The skilled person is aware of methods foraccomplishing C-terminal modification, such as by different types ofpre-made matrices for peptide synthesis.

In another embodiment, the additional amino acid residues comprise acysteine residue at the N- and/or C-terminal of the polypeptide. Such acysteine residue may directly precede and/or follow the amino acidsequence as defined in (i) or (iii) or may precede and/or follow anyother additional amino acid residues as described above. Examples ofalbumin binding polypeptides comprising a cysteine residue at the N-and/or C-terminal of the polypeptide chain are set out in SEQ IDNO:449-450 (C-terminal) and SEQ ID NO:451-452 (N-terminal). By theaddition of a cysteine residue to the polypeptide chain, a thiol groupfor site directed conjugation of the albumin binding polypeptide may beobtained. Alternatively, a selenocysteine residue may be introduced atthe C-terminal of the polypeptide chain, in a similar fashion as for theintroduction of a cysteine residue, to facilitate site-specificconjugation (Cheng et al, Nat Prot 1:2, 2006).

In one embodiment, the albumin binding polypeptide comprises no morethan two cysteine residues. In another embodiment, the albumin bindingpolypeptide comprises no more than one cysteine residue.

In another embodiment, the additional amino acid residues of the albuminbinding polypeptide comprise a “tag” for purification or detection ofthe polypeptide, such as a hexahistidyl (His₆) tag, (SEQ ID NO: 34), ora “myc” (“c-Myc”) tag or a “FLAG” tag for interaction with antibodiesspecific to the tag and/or to be used in purification. The skilledperson is aware of other alternatives.

Because of the presence of an albumin binding motif, the ABD peptidebinds to albumin with a K value of the interaction that is at most1×10⁻⁶ M and even more preferably at most 1×10⁻⁹ M (even tighteraffinity). More preferably the K value of the interaction that is atmost 1×10⁻¹⁰ M, even more preferably is at most 1×10⁻¹¹ M, yet even morepreferably is at most 1×10⁻¹² M, and even further is at most 1×10⁻¹³ M.

Sequences of individual albumin binding domain polypeptides suitable forfusion with the active hormone domain peptides as described herein aresuperior to those presented in Jonsson et al. (Id.) and in WO09/016043.Selected compounds are disclosed in Table 1 below. See also PCTPublished Appl. No. WO 2012/004384. Also encompassed by the presentinvention is an albumin binding polypeptide having an amino acidsequence with 95% or greater identity to a sequence selected from SEQ IDNO:301-452, SEQ ID NO:454-463, and SEQ ID NO:500-593. In particularembodiments, the sequence of the albumin binding polypeptide is selectedfrom SEQ ID NO:313, SEQ ID NO:448, SEQ ID NO:463, SEQ ID NO:500, SEQ IDNO:501, SEQ ID NO:502, and sequences having 95% or greater identitythereto. In other embodiments of the invention, the sequence of thealbumin binding polypeptide is selected from SEQ ID NO:454-463 andsequences having 95% or greater identity thereto. In yet furtherembodiments, the sequence of the albumin binding polypeptide is selectedfrom SEQ ID NO:500-593 and sequences having 95% or greater identitythereto.

Exemplary ABD species include, but are not limited to, the compounds setforth in Table 1 following and the Examples.

TABLE 1 Selected ABD peptides SEQ ID Designation ABD peptide sequenceNO: PEP07271 GSSLASAKEAANAELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 455PEP07554 GSSLASAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 456 PEP07912GLASAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 457 PEP07914GLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 458 PEP07911GLASAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 459 PEP07834ALASAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 460 PEP07844GSSLASAKEAANAELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 461 PEP07983GSLASAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 462 PEP07986GSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 463 PEP08185GSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG 448LAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 313SLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 500LAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG 501SLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG 502LAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 503SLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 504GSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 505LAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 506SLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 507GSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 508LAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 509SLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 510GSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 511LAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 512SLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 513GSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 514LAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 515SLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 516GSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 517LAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 518SLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 519GSLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 520LAEAKEAANRELDAYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 521SLAEAKEAANRELDAYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 522GSLAEAKEAANRELDAYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 523LAQAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 524SLAQAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 525GSLAQAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 526LAEAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 527SLAEAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 528GSLAEAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 529LAQAKEAANAELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 530SLAQAKEAANAELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 531GSLAQAKEAANAELDSYGVSDFYKRLIEKAKTVEGVEALKDAILAALP 532LAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 533SLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 534GSLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 535LAEAKEAANRELDAYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 536SLAEAKEAANRELDAYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 537GSLAEAKEAANRELDAYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 538LAQAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 539SLAQAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 540GSLAQAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 541LAEAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 542SLAEAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 543GSLAEAKEAANRELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 544LAQAKEAANAELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 545SLAQAKEAANAELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 546GSLAQAKEAANAELDSYGVSDFYKRLIEKAKTVEGVEALKEAILAALP 547LAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 548SLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 549GSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 550LAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 551SLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 552GSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 553LAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 554SLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 555GSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 556LAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 557SLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 558GSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 559LAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 560SLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 561GSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILASLP 562LAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 563SLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 564GSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 565LAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 566SLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 567GSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 568LAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 569SLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 570GSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 571LAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 572SLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 573GSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 574LAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 575SLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 576 GSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAELP 577LAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 578SLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 579GSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 580LAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 581SLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 582GSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 583LAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 584SLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 585GSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 586LAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 587SLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 588GSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 589LAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 590SLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 591GSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILKALP 592LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP 593

In preferred engineered polypeptide embodiments the ABD comprisesLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP (SEQ ID NO: 313), and itsN-terminally extended ABD sequence forms includingSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP (SEQ ID NO: 500) andGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP (SEQ ID NO:463;PEP07986). The serine in position 2 is capping the sequence, raising Tmapproximately 2° C. compared to having a glycine or an alanine in thisposition.

In preferred engineered polypeptide embodiments where Cys-conjugation isdesired the preferred ABD comprisesLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG (SEQ ID NO: 501) and itsN-terminally extended ABD sequence forms includingSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG (SEQ ID NO: 502) andGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG (SEQ ID NO: 448;PEP08185).

In one embodiment of the engineered polypeptides described herein,particularly those ending at its C-terminus with proline or other aminoacid known to racemize during peptide synthesis, a glycine can be addedto the C-terminus to counter potential problems with racemization of theC-terminal amino acid residue. Alternatively the C-terminal amino acidcan in its (alpha-amino group) amidated form, e.g. proline versusproline amide, rather than ending with a glycine. However, if theamidated polypeptide is desired to be produced by recombinant ratherthan chemical synthesis, then amidation of the C-terminal amino acid canbe performed by several methods known in the art, e.g. use of amidatingPAM enzyme.

The ABD herein fold completely reversibly, that is they can be denaturedand will refold spontaneously to the desired active tertiary structure.This was assessed by circular dichroism spectra analysis, for example ofABD SEQ ID NO:463, where one compares spectrum taken at 20° C. (foldedstate) and a second spectrum taken after heating to 90° C. (heatdenaturation) a third spectrum taken following return to 20° C.(refolded state). During this procedure the Tm can be determined.

Another aspect of the engineered polypeptides is that the ABD canprovide an increase in the solubility in aqueous solution of a poor orlow soluble leptin variant. This property can be imparted by the ABDitself or because of the ensuing complex of the engineered polypeptidebound to highly soluble albumin in vivo or in vitro, which associationincreases the solubility of the engineered polypeptide in aqueoussolution. Thus, in an embodiment of this further aspect, there isprovided a composition, comprising a leptin or leptin analog compoundwhich per se has a solubility in water of no more than 1 mg/ml, or nomore than 2 mg//ml or no more than 5 g/ml, covalently coupled to analbumin binding domain as a fusion protein or conjugate as describedherein, wherein the compound and the albumin binding polypeptide, fusionprotein or conjugate are covalently coupled and the solubility of theengineered polypeptide is greater than that of the unfused (or notconjugated) native leptin or leptin analog compound.

Binding to Albumin.

Serum albumin is the most abundant protein in mammalian sera (40 g/L;approximately 0.7 mM in humans) where it binds a variety of moleculesincluding but not limited to lipids and bilirubin (Peters T, 1985,Advances in Protein Chemistry 37:161). It has been observed that thehalf-life of serum albumin is directly proportional to the size of theanimal, where for example human serum albumin (HSA) has a half-life of19 days and rabbit serum albumin has a half-life of about 5 days(McCurdy T R et al., J. Lab. Clin. Med. 143:115, 2004). Human serumalbumin is widely distributed throughout the body, in particular in theintestinal and blood compartments, where it is mainly involved in themaintenance of osmolarity. Structurally, albumins are single-chainproteins comprising three homologous domains and totaling 584 or 585amino acids (Dugaiczyk L et al., 1982, Proc. Natl. Acad. Sci. USA79:71). Albumins contain 17 disulfide bridges and a single reactivethiol, C34, but lack N-linked and O-linked carbohydrate moieties(Peters, 1985, Id.; Nicholson J P et al., 2000, Br J Anaesth 85:599).The lack of glycosylation simplifies recombinant expression of albumin.This property of albumin, together with the fact that itsthree-dimensional structure is known (He, X M and Carter, D C, Nature358:209 1992), has made it an attractive candidate for use inrecombinant fusion proteins. Such fusion proteins generally combine atherapeutic protein (which would be rapidly cleared from the body uponadministration of the protein per se) and a plasma protein (whichexhibits a natural slow clearance) in a single polypeptide chain. Seee.g., Sheffield W P, 2001, Curr. Drug Targets Cardiovacs. Haematol.Disord. 1:1). Such proteins may provide clinical benefits in requiringless frequent injection and higher levels of therapeutic protein invivo. However, the engineered polypeptides herein are not conjugated toalbumin, but instead contain motifs that allow non-covalent binding toalbumin.

Albumin Half-Life.

It has been observed that the half-life of albumin in different speciesgenerally adheres to allometric scaling based on animal weight. Forexample, the albumin half-life in mouse, rat, rabbit and human has beenestimated as 1, 1.9, 5.6 and 19 days, respectively. Indeed, powerfitting analysis (Davies & Morris, 1993, Pharm. Res. (N.Y.)10:1093-1095) provides the equation:

Albumin half-life(days)=3.75*body weight(kg)^(0.368).

Further Embodiments

It is understood that each of the polypeptides disclosed herein are alsocontemplated to include (optionally) a methionine at the N-terminus inframe with the naturally-occurring first amino acid thereof. Forexample, metreleptin (leptin A100) consists of mature human leptin towhich has been added an N-terminal methionine, as disclosed in SEQ IDNO:20. Similarly, a methionine residue may be included at the N-terminusof any of the amino acid sequences and Formulae disclosed hereinthroughout. It is further understood that where a C-terminal Gly appearsin an engineered polypeptide sequence set forth herein, the residue maybe lost during subsequent amidation.

In some embodiments, a leptin, a leptin analog, a leptin activefragment, or a leptin derivative can have at least 50%, for example 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher,sequence identity relative to a parent leptin. In some embodiments, theparent leptin is a leptin set out in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, IDNO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669,SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ IDNO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677 or SEQ ID NO:680.Accordingly, in some embodiments, a leptin, a leptin analog, a leptinactive fragment, or a leptin derivative may have at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or evenhigher, sequence identity relative to any leptin selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, and SEQ ID NO:23. In some embodiments, a leptin, a leptin analog,a leptin active fragment, or a leptin derivative may have at least 50%,for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% oreven higher, sequence identity relative to the leptin set forth in SEQID NO:20. In some embodiments, a leptin, a leptin analog, a leptinactive fragment, or a leptin derivative may have at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or evenhigher, sequence identity relative to any leptin selected from the groupconsisting SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO:27, SEQID NO: 28, or SEQ ID NO:29. In some embodiments, a leptin analog mayhave at least 90% sequence identity relative to the leptin set forth inSEQ ID NO:20. In some embodiments, a leptin analog may have at least 50%sequence identity relative to the leptin set forth in SEQ ID NO:1, SEQID NO:2, ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673,SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677 or SEQ IDNO:680. In some embodiments, a leptin analog may have at least 90%sequence identity relative to the leptin set forth in SEQ ID NO:1, SEQID NO: 2, ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673,SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677 or SEQ IDNO:680. In some embodiments, a leptin analog may have at least 50%sequence identity relative to the leptin set forth in SEQ ID NO:14 orSEQ ID NO:15. In some embodiments, a leptin analog may have at least 90%sequence identity relative to the leptin set forth in SEQ ID NO:14 orSEQ ID NO:15. In some embodiments, a leptin analog may have at least 50%sequence identity relative to the leptin set forth in SEQ ID NO: 32. Insome embodiments, a leptin analog may have at least 90% sequenceidentity relative to the leptin set forth in SEQ ID NO:32. In someembodiments, a leptin analog may have at least 50% sequence identityrelative to the leptin set forth in SEQ ID NO: 33. In some embodiments,a leptin analog may have at least 90% sequence identity relative to theleptin set forth in SEQ ID NO:33. In some embodiments, a leptin analogmay have at least 50% sequence identity relative to the leptin set forthin SEQ ID NO:10 or SEQ ID NO:11. In some embodiments, a leptin analogmay have at least 90% sequence identity relative to the leptin set forthin SEQ ID NO:10 or SEQ ID NO:11. In some embodiments, a leptin analogmay have at least 50% sequence identity relative to the leptin set forthin SEQ ID NO:12 or SEQ ID NO:13. In some embodiments, a leptin analogmay have at least 90% sequence identity relative to the leptin set forthin SEQ ID NO:12 or SEQ ID NO:13. Additionally, leptins may be designed,prepared, and used in accordance with the invention in which 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even 21amino acids of a leptin selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ IDNO:146, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672,SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ IDNO:677, and SEQ ID NO:680; is/are substituted with another amino acid,such as a conservative amino acid or a non-conservative amino acid, oris/are otherwise altered. As customary in the art, the term“conservative” in the context of amino acid substitutions refers tosubstitution which maintains properties of charge type (e.g., anionic,cationic, neutral, polar and the like), hydrophobicity orhydrophilicity, bulk (e.g., van der Waals contacts and the like), and/orfunctionality (e.g., hydroxy, amine, sulhydryl and the like). The term“non-conservative” refers to an amino acid substitution which is notconservative.

Additionally, as is understood in the art, for example, murine leptins,rat leptins, bovine leptins, porcine leptins, and rhesus monkey leptins,such as those disclosed herein, are each substantially homologous tohuman leptins; in particular, the mature forms of these leptins aresubstantially homologous to mature leptins, and further, particularlynear the N-terminal portion of the protein. One may prepare analogs ofsuch leptins, such as mature human leptin form 1 (SEQ ID NO:16) andmetreleptin (SEQ ID NO:20), such as by substituting or otherwisealtering amino acid residues at one or more positions in such sequenceswhere divergence is observed in a corresponding mature mouse, rat,bovine, porcine, or rhesus monkey leptin. For example, mature humanleptins (e.g., SEQ ID NO:20) elicits biological responses in, forexample, mice, rat, and monkey). See, e.g., WO 98/28427, WO 2009/064298,US2007/0020284, US2008/0207512, and Murakami et al., 1995, Biochem.Biophys. Res. Comm. 209: 944-952. Because human mature leptins havebiological activity in, e.g., such species, leptins may be designed andprepared in which one or more amino acids at positions which aredivergent at the corresponding position(s) in a leptin from one or moreof such species are substituted with the amino acid(s) at suchcorresponding divergent positions.

For example, using a human mature leptin protein according to SEQ IDNO:16 wherein the first amino acid is valine and the amino acid atposition 146 is cysteine, one may substitute with another amino acid oneor more of the amino acids at positions 32, 35, 50, 64, 68, 71, 74, 77,89, 97, 100, 101, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and145 with the corresponding amino acid(s) found at the correspondingposition(s) in SEQ ID NO:143) in order to design, prepare, and useengineered polypeptides in accordance with the invention. Additionally,one may also substitute another amino acid, such as a conservative aminoacid or a non-conservative amino acid, into one or more of positions 32,35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, 111,112, 118, 136, 138, 142, and 145 of, for example, SEQ ID NO:16 in orderto design, prepare, and use engineered polypeptides in accordance withthe invention.

One may further prepare additional leptins based on the mature ratleptin protein sequence (SEQ ID NO:12). See, e.g., WO 98/28427,US2007/0020284, and Murakami et al., 1995, Id., herein incorporated byreference in their entireties and for all purposes. Mature rat leptindiffers from mature human leptin form 1 (SEQ ID NO:16) at the followingpositions: 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102,105, 106, 107, 108, 111, 112, 118, 136, 138 and 145. Accordingly, at oneor more of such positions in SEQ ID NO:16, one may substitute the aminoacid found at the corresponding position(s) found in mature rat leptin(SEQ ID NO:12) in order to design, prepare, and use engineeredpolypeptides in accordance with the invention. Additionally, one mayalso substitute another amino acid, such as a conservative amino acid ora non-conservative amino acid, into one or more of positions 4, 32, 33,35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108,111, 112, 118, 136, 138 and 145 of, for example, SEQ ID NO:16, in orderto design, prepare, and use engineered polypeptides in accordance withthe invention.

The positions from both mature rat leptin (SEQ ID NO:12) and maturemurine leptin form 1 (SEQ ID NO:143) which diverge from the mature humanleptin form 1 (SEQ ID NO:16) are: 4, 32, 33, 35, 50, 64, 68, 71, 74, 77,78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142,and 145. Accordingly, at one or more of such positions in SEQ ID NO:16,one may substitute the amino acid found at the corresponding position(s)found in mature rat leptin sequence (SEQ ID NO:12) or mature murine form1 sequence (SEQ ID NO:143) in order to design, prepare, and useengineered polypeptides in accordance with the invention. Additionally,one may also substitute another amino acid, such as a conservative aminoacid or a non-conservative amino acid, into one or more of positions 4,32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107,108, 111, 112, 118, 136, 138, 142, and 145 in order to design, prepare,and use engineered polypeptides in accordance with the invention.

In addition, the amino acids found in rhesus monkey mature leptin (SEQID NO:10) which diverge from mature human leptin form 1 (SEQ ID NO:16)are (with amino acid residues noted in parentheses in one letter aminoacid abbreviation): 8 (S), 35 (R), 48(V), 53(Q), 60(I), 66(I), 67(N),68((L), 89(L), 100(L), 108(E), 112 (D), and 118 (L). Since human matureleptins elicit biological response monkeys, a leptin, such as maturehuman leptin form 1 (SEQ ID NO:16) having one or more of the rhesusmonkey divergent amino acids replaced with another amino acid, such asthe amino acids in parentheses, may be employed in designing, preparing,and using engineered polypeptides in accordance with the invention. Itshould be noted that certain rhesus divergent amino acids are also thosefound in, for example, the above mature murine leptin form 1 (positions35, 68, 89, 100 and 112). Thus, one may prepare leptins in which one ormore amino acids at positions 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66,67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112,118, 136, 138, 142, and 145 of, e.g., mature human leptin form 1 (SEQ IDNO:16) are replaced by the corresponding amino acid(s) at suchposition(s) in murine or rhesus monkey leptins (e.g., SEQ ID NO:143and/or SEQ ID NO:10).

Other leptins may be prepared by deleting a part of a leptin amino acidsequence, provided that such a leptin amino acid sequence may elicit abiological response. Such leptin amino acid sequences are leptin activefragments. For example, mature murine leptins, mature rhesus monkeyleptins, mature human leptins, and mature rat leptins, and other leptinsall lack the N-terminal 21 amino acid signal sequence that is present inthe unprocessed, full-length forms of such leptin.

One may prepare the following active leptin fragments of such matureleptins:

(a) amino acids 98-146

(b) amino acids 1-32

(c) amino acids 40-116

(d) amino acids 1-99 and (connected to) 112-146

(e) amino acids 1-99 and (connected to) 112-146 having one or more ofamino acids 100-111 placed between amino acids 99 and 112.

In addition, such active leptin fragments may also be prepared in whichone or more of the amino acids at positions in, e.g., mature humanleptin form 1 that are substituted with the amino acids found at thecorresponding position(s) found in, e.g., rat, murine, monkey, porcine,and/or bovine mature leptins as disclosed above. Furthermore, anysubstitutions or alterations may be in the form of altered amino acids,such as peptidomimetics or D-amino acids.

Additionally, the present invention encompasses engineered polypeptideswhich comprise a leptin, a leptin analog, a leptin active fragment, or aleptin derivative as described above, wherein the leptin, a leptinanalog, a leptin active fragment, or a leptin derivative is selectedfrom:

(a) the amino acid sequence 1-146 of a leptin selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ IDNO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ ID NO:665, SEQID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670,SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ IDNO:675, SEQ ID NO:676, and SEQ ID NO:677; in which a different aminoacid is substituted in one or more of the following positions andretaining the same numbering (even in the absence of a glutaminylresidue at position 28): 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89,97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145;

(b) the amino acid sequence of subpart (a) in which the glutaminylresidue at position 28 is absent;

(c) the amino acid sequence of subparts (a) or (b) in which a methionylresidue is added at the N-terminus;

(d) a leptin consisting of a fragment of the amino acid sequence of (a),(b), or (c) selected from the group consisting of:

-   -   (i) amino acids 98-146    -   (ii) amino acids 1-32    -   (iii) amino acids 40-116    -   (iv) amino acids 1-99 and 112-146    -   (v) amino acids 1-99 and 112-146 in which one or more of amino        acids 100-111 is placed between amino acids 99 and 112; and,    -   (vi) the amino acid sequence of subpart (i) wherein one or more        of amino acids 100, 102, 105, 106, 107, 108, 111, 118, 136, 138,        142, and 145 is substituted with another amino acid;    -   (vii) the amino acid sequence of subpart (ii) wherein one or        more of amino acids 4, 8 and 32 is substituted with another        amino acid;    -   (viii) the amino acid sequence of subpart (iii) wherein one or        more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78,        89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 is replaced        with another amino acid;    -   (ix) the amino acid sequence of subpart (iv) wherein one or more        of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68,        71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 is        replaced with another amino acid; and    -   (x) the amino acid sequence of subpart (v) wherein one or more        of amino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89,        97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and        145 is replaced with another amino acid;    -   (xi) the leptin of any of subparts (i)-(x) wherein a methionine        has been added at the N-terminus; and

(e) the leptin of any of subparts (a) through (e) to which a chemicalmoiety is attached;

(f) the leptin of subpart (g) wherein said chemical moiety is a watersoluble polymer moiety;

(g) a leptin of subpart (f) wherein said water soluble polymer moiety ispolyethylene glycol;

(h) a leptin of subpart (f) wherein said water soluble polymer moiety isa polyaminoacid moiety; and

(i) a leptin of any one of subparts (e) through (h) wherein said moietyis attached at solely the N-terminus of said protein moiety.

With regard to the above, leptins to which a chemical moiety is attachedare leptin derivatives. Derivatization of leptins by attachment of oneor more chemical moieties has been found to provide some advantage undercertain circumstances, such as increasing the stability and circulationtime of the therapeutic protein and decreasing immunogenicity andpropensity for, for example, generation of neutralizing antibodiesand/or incidence of injection site reactions. See, e.g., WO 98/28427,US2007/0020284, U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18,1979. For a review, see Abuchowski et al., in ENZYMES AS DRUGS. (J. S.Holcerberg and J. Roberts, eds. pp. 367-383 (1981)); Francis et al., Id.Accordingly, when employing a derivatized leptin and an ABM or an ABD,one may advantageously generate engineered polypeptides of the inventionpossessing advantages provided by both entities.

Leptin derivatives may constitute leptins to which a chemicalmodification has been made of one or more of its amino acid side groups,α-carbon atoms, terminal amino group, or terminal carboxylic acid group.A chemical modification includes, but is not limited to, attaching oneor more chemical moieties, creating new bonds, and removing one or morechemical moieties. Modifications at amino acid side groups include,without limitation, alkylation, acylation, ester formation, amideformation, maleimide coupling, acylation of lysine ε-amino groups,N-alkylation of arginine, histidine, or lysine, alkylation of glutamicor aspartic carboxylic acid groups, and deamidation of glutamine orasparagine. Modifications of the terminal amino include, withoutlimitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal amino include, withoutlimitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications, such as alkylacyls, branched alkylacyls, alkylaryl-acyls.Modifications of the terminal carboxy group include, without limitation,the amide, lower alkyl amide, dialkyl amide, arylamide, alkylarylamideand lower alkyl ester modifications. Lower alkyl is C₁-C₄ alkyl.Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled syntheticchemist. The α-carbon of an amino acid may be mono- or dimethylated.

Such derivatives include leptins conjugated to one or more water solublepolymer molecules, such as polyethylene glycol (“PEG”) or fatty acidchains of various lengths (e.g., stearyl, palmitoyl, octanoyl), by theaddition of polyamino acids, such as poly-his, poly-arg, poly-lys, andpoly-ala, or by addition of small molecule substituents include shortalkyls and constrained alkyls (e.g., branched, cyclic, fused,adamantyl), and aromatic groups. In some embodiments, the water solublepolymer molecules will have a molecular weight ranging from about 500Daltons to about 60,000 Daltons.

Such polymer-conjugations may occur singularly at the N- or C-terminusor at the side chains of amino acid residues within the sequence of aleptin as disclosed herein. Alternatively, there may be multiple sitesof derivatization along the amino acid sequence of such a leptin.Substitution of one or more amino acids with lysine, aspartic acid,glutamic acid, or cysteine may provide additional sites forderivatization. See, e.g., U.S. Pat. Nos. 5,824,784 and 5,824,778. Insome embodiments, a leptin may be conjugated to one, two, or threepolymer molecules.

In some embodiments, the water soluble polymer molecules are linked toan amino, carboxyl, or thiol group, and may be linked by N or C termini,or at the side chains of lysine, aspartic acid, glutamic acid, orcysteine. Alternatively, the water soluble polymer molecules may belinked with diamine and dicarboxylic groups. In some embodiments, aleptin is conjugated to one, two, or three PEG molecules through anepsilon amino group on a lysine amino acid.

Leptin derivatives also include leptins with chemical alterations to oneor more amino acid residues. Such chemical alterations includeamidation, glycosylation, acylation, sulfation, phosphorylation,acetylation, and cyclization. The chemical alterations may occursingularly at the N- or C-terminus or at the side chains of amino acidresidues within the sequence of a leptin. In one embodiment, theC-terminus of these peptides may have a free —OH or —NH₂ group. Inanother embodiment, the N-terminal end may be capped with anisobutyloxycarbonyl group, an isopropyloxycarbonyl group, ann-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group(“isocap”), an octanyl group, an octyl glycine group (denoted as“G(Oct)” or “octylGly”), an 8-aminooctanic acid group, a dansyl, and/ora Fmoc group. In some embodiments, cyclization can be through theformation of disulfide bridges. Alternatively, there may be multiplesites of chemical alteration along the leptin amino acid sequence.

In certain embodiments, leptins are chemically altered to include aBolton-Hunter group. Bolton-Hunter reagents are known in the art(“Radioimmunoassay and related methods,” A. E. Bolton and W. M. Hunter,Chapter 26 of HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUME I,IMMUNOCHEMISTRY, edited by D. M. Weir, Blackwell ScientificPublications, 1986), and may be used to introduce tyrosine-like moietieswith a neutral linkage, through amino-terminal α-amino groups or ε-aminogroups of lysine. In some embodiments, the N-terminal end of a leptin ismodified with a Bolton-Hunter group. In some embodiments, an internallysine residue is modified with a Bolton-Hunter group. In someembodiments, there may be multiple sites of Bolton-Hunter modificationalong the leptin amino acid sequence. Bolton-Hunter reagents used forpolypeptide modification are commercially available, and may include,but are not limited to, water-soluble Bolton-Hunter reagent,Sulfosuccinimidyl-3-[4-hydrophenyl]propionate (Pierce Biotechnology,Inc., Rockford, Ill.) and Bolton-Hunter reagent-2, N-Succinimidyl3-(4-hydroxy-3-iodophenyl) Priopionate (Wako Pure Chemical Industries,Ltd., Japan, catalog #199-09341). An exemplary Bolton-Hunter groupconjugated through an amide linkage to a leptin is illustrated below,wherein the dashed line passes through the amide bond:

Leptins may be iodinated (such as radiolabeled with ¹²⁵I) before orafter Bolton-Hunter modification.

In order to prepare engineered polypeptides in accordance with theinvention, a leptin derivative for use in the preparation of such mayinclude one or more modifications of a “non-essential” amino acidresidue. In the context of the invention, a “non-essential” amino acidresidue is a residue that can be altered, e.g., derivatized, withoutabolishing or substantially reducing the activity (e.g., the agonistactivity) of the leptin. The engineered polypeptides of the inventionmay include derivatizations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acid residues of the leptin moiety; of these, one or more aminoacid residues may be non-essential amino acid residues. Additionally,the polypeptides of the invention may be derivatized such that theyinclude additions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acids of the leptin moiety without abolishing or substantiallyreducing the activity of the polypeptide. Additionally, suchnon-essential amino acid residues may be substituted with an amino acidresidue that is amenable to derivatization as described throughout.

As used throughout, “amino acid,” “amino acid residue” and the likerefer to natural amino acids, unnatural amino acids, and modified aminoacids. Unless stated to the contrary, any reference to an amino acid,generally or specifically by name, includes reference to both the D andthe L stereoisomers if their structure allow such stereoisomeric forms.Natural amino acids include alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr)and valine (Val). Unnatural amino acids include, but are not limited tohomolysine, homoarginine, homoserine, azetidinecarboxylic acid,2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionicacid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid,2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid,desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine,allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine,allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.Additional unnatural amino acids include modified amino acid residueswhich are chemically blocked, reversibly or irreversibly, or chemicallymodified on their N-terminal amino group or their side chain groups, asfor example, N-methylated D and L amino acids or residues wherein theside chain functional groups are chemically modified to anotherfunctional group. For example, modified amino acids include methioninesulfoxide; methionine sulfone; aspartic acid-(beta-methyl ester), amodified amino acid of aspartic acid; N-ethylglycine, a modified aminoacid of glycine; or alanine carboxamide, a modified amino acid ofalanine. Additional residues that can be incorporated are described inSandberg et al., J. Med. Chem. 41: 2481-91, 1998.

As mentioned above, chemical moieties suitable for such derivatizationof leptins and other polypeptides include, for example, various watersoluble polymers. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer/protein conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the engineeredpolypeptides and leptins, the effectiveness of the derivatization may beascertained by administering the derivatized leptin or the derivatizedengineered polypeptide, in the desired form (i.e., by osmotic pump, or,more preferably, by injection or infusion, or, further formulated fororal, pulmonary or nasal delivery, for example), and observingbiological effects and biological responses as described herein.

Such a water soluble polymer may be selected from the group consistingof, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrolidone)polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyolsand polyvinyl alcohol. Polyethylene glycol propionaldehyde may haveadvantages in manufacturing due to its stability in water. Also,succinate, styrene, and hydroxyethyl starch may also be used.

Leptin derivatives used in the design and preparation of engineeredpolypeptides in accordance with the invention may be prepared byattaching polyaminoacids or branch point amino acids to the leptinmoiety. For example, the polyaminoacid may be an additional carrierprotein, such as an Fc moiety, which can serve to also increase thecirculation half life of the leptin or the engineered polypeptide, inaddition to the advantages achieved via attachment of an ABM or an ABD.Additionally, such polyaminoacids may be selected from the groupconsisting of serum albumin (such as human serum albumin), an additionalantibody or portion thereof (e.g. the Fc region), or otherpolyaminoacids, e.g. polylysines. As indicated below, the location ofattachment of the polyaminoacid may be at the N-terminus of the leptinmoiety, or C-terminus, or other places in between, and also may beconnected by a chemical “linker” moiety to the leptin, such as apeptidic linker or a non-peptidic linker.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kilodaltons (kDa) and about 100 kDa (the term “about”indicating that in preparations of polyethylene glycol, some moleculeswill weigh more, some less, than the stated molecular weight) for easein handling and manufacturing. In certain embodiments, the polyethyleneglycol is between about 2 kDa and about 60 kDa. In certain embodiments,the polyethylene glycol is between about 2 kDa and about 40 kDa. Incertain embodiments, the polyethylene glycol is between about 5 kDa andabout 40 kDa. In certain embodiments, the polyethylene glycol is betweenabout 10 kDa and about 40 kDa. In certain embodiments, the polyethyleneglycol is between about 5 kDa and about 30 kDa. In certain embodiments,the polyethylene glycol is between about 5 kDa and about 20 kDa. Incertain embodiments, the polyethylene glycol is between about 10 kDa andabout 20 kDa. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,solubility characteristics, the effects, if any, on biological activity,the ease in handling, the degree or lack of antigenicity and other knowneffects of the polyethylene glycol attached to a leptin and/or to anengineered polypeptide of the invention). Additional considerations thatmay influence the selection of a PEG of a particular molecular weightwhich may be attached to a leptin to generate a leptin derivative inaccordance with the invention include the extent to which such amolecular weight PEG may: mitigate aggregation and/or increase thesolubility of the leptin and/or the engineered polypeptide, when presentin a pharmaceutically acceptable composition or formulation, or whenexposed to physiological fluids or tissues upon administration to asubject (such as by injection); mitigate the incidence of injection sitereactions caused by administration of the leptin or the engineeredpolypeptide upon administration to a subject by injection; mitigate thegeneration of neutralizing antibodies that may be raised against theleptin or the engineered polypeptide as a result of administration ofsuch a leptin or an engineered polypeptide to a subject; and the like.

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the resultant effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to leptin molecules orengineered polypeptide molecules to be derivatized will vary, as willtheir concentrations in the reaction mixture. In general, the optimumratio, in terms of efficiency of reaction in that there is no excessunreacted leptin (or engineered polypeptide, as the case may be) orpolymer, will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

The chemical moieties should be attached to the leptin and/or theengineered polypeptide with consideration of the effects on functionalor antigenic domains of the leptin and/or to the engineered polypeptide.There are a number of attachment methods available to those skilled inthe art. E.g., EP 0 401 384 herein incorporated by reference (couplingPEG to G-CSF), see also Malik et al., 1992, Exp. Hematol. 20:1028-1035(reporting pegylation of GM-C SF using tresyl chloride). For example,polyethylene glycol may be covalently bound through amino acid residuesvia a reactive group, such as, a free amino or carboxyl group. Reactivegroups are those to which an activated polyethylene glycol molecule maybe bound. The amino acid residues having a free amino group may includelysine residues and the N-terminal amino acid residue. Those having afree carboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecule(s). Preferred for therapeutic purposes is attachment at anamino group, such as attachment at the N-terminus or lysine group.Attachment at residues important for receptor binding should be avoidedif receptor binding is desired.

In certain embodiments the 1 to 30 or less amino acids are selected fromglycine, alanine, proline, asparagine, glutamine, lysine, aspartate, andglutamate. In a further embodiment the receptor binding is desired.

One may specifically desire to design and prepare an N-terminallychemically modified leptin for use in the preparation of engineeredpolypeptides of the invention. Using polyethylene glycol as anillustration of the present compositions, one may select from a varietyof polyethylene glycol molecules (by molecular weight, branching, etc.),the proportion of polyethylene glycol molecules to leptin or engineeredpolypeptide molecules, as the case may be, in the reaction mix, the typeof pegylation reaction to be performed, and the method of obtaining theselected N-terminally pegylated protein. The method of obtaining theN-terminally pegylated preparation (i.e., separating this moiety fromother monopegylated moieties if necessary) may be by purification of theN-terminally pegylated material from a population of pegylated proteinmolecules. Selective N-terminal chemical modification may beaccomplished by reductive alkylation which exploits differentialreactivity of different types of primary amino groups (lysine versus theN-terminal) available for derivatization in a particular protein. Underthe appropriate reaction conditions, substantially selectivederivatization of the protein at the N-terminus with a carbonyl groupcontaining polymer is achieved. For example, one may selectivelyN-terminally pegylate the protein by performing the reaction at a pHwhich allows one to take advantage of the pK_(a) differences between theε-amino group of the lysine residues and that of the α-amino group ofthe N-terminal residue of the protein. By such selective derivatization,attachment of a water soluble polymer to a protein is controlled: theconjugation with the polymer takes place predominantly at the N-terminusof the protein and no significant modification of other reactive groups,such as the lysine side chain amino groups, occurs. Using reductivealkylation, the water soluble polymer may be of the type describedabove, and should have a single reactive aldehyde for coupling to theprotein. Polyethylene glycol propionaldehyde, containing a singlereactive aldehyde, may be used.

In some embodiments, compounds are provided having a linker, for exampleL1, as described herein, covalently linking a polypeptide hormone domainwith an ABD peptide. In some embodiments, a first linker (L1) covalentlylinks HD1 within the engineered polypeptide. In some embodiments, thepolypeptide hormone domain (e.g., HD1) as described herein) can becovalently linked to the ABD peptide via a peptide linker. Any linker isoptional; i.e., any linker may simply be a bond. When present thechemical structure of a linker is not critical because it serves mainlya spacer function. In one embodiment the linker comprises from 1 to 30or less amino acids linked by peptide bonds. The amino acids can beselected from the 20 naturally occurring amino acids. Alternatively,non-natural amino acids can be incorporated either by chemicalsynthesis, post-translational chemical modification or by in vivoincorporation by recombinant expression in a host cell. Some of theseamino acids may be glycosylated.

In certain embodiments the 1 to 30 or less amino acids are selected fromglycine, alanine, proline, asparagine, glutamine, lysine, aspartate, andglutamate. In a further embodiment the linker is made up of a majorityof amino acids that are sterically unhindered, such as glycine, alanineand/or serine. Polyglycines are particularly useful, e.g. (Gly)₃, (Gly)₄(SEQ ID NO:116), (Gly)₅ (SEQ ID NO:117), as are polyalanines,poly(Gly-Ala), poly(Glyn-Ser), poly (Gly_(n)-Glu), poly(Gly_(n)-Lys),poly(Gly_(n)-Asp), and poly(Gly_(n)-Arg) motifs. Other specific examplesof linkers are (Gly)₃Lys(Gly)₄ (SEQ ID NO:118); (Gly)₃AsnGlySer(Gly)₂(SEQ ID NO:119); (Gly)₃Cys(Gly)₄ (SEQ ID NO:120); and GlyProAsnGlyGly(SEQ ID NO:121). Combinations of Gly and Ala are particularly useful asare combination of Gly and Ser. Thus, in a further embodiment thepeptide linker is selected from the group consisting of a glycine richpeptide, e.g. Gly-Gly-Gly; the sequences [Gly-Ser]_(n) (SEQ ID NO:122),[Gly-Gly-Ser]_(n) (SEQ ID NO:123), [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO:124)and [Gly-Gly-Gly-Gly-Ser]₁ (SEQ ID NO:125), where n is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, for example, [Gly-Gly-Gly Ser]₁ (SEQ ID NO: 149),[Gly-Gly-Gly-Gly Ser]₁ (SEQ ID NO: 150), [Gly-Gly-Gly Ser]_(n) (SEQ IDNO: 151), or [Gly-Gly-Gly-Gly Ser]_(n) (SEQ ID NO: 152).

In certain embodiments, charged linkers may be used. Such chargeslinkers may contain a significant number of acidic residues (e.g., Asp,Glu, and the like), or may contain a significant number of basisresidues (e.g., Lys, Arg, and the like), such that the linker has a pIlower than 7 or greater than 7, respectively. As understood by theartisan, and all other things being equal, the greater the relativeamount of acidic or basic residues in a given linker, the lower orhigher, respectively, the pI of the linker will be. Such linkers mayimpart advantages to the engineered polypeptides disclosed herein, suchas improving solubility and/or stability characteristics of suchpolypeptides at a particular pH, such as a physiological pH (e.g.,between pH 7.2 and pH 7.6, inclusive), or a pH of a pharmaceuticalcomposition comprising such polypeptides.

For example, an “acidic linker” is a linker that has a pI of less than7; between 6 and 7, inclusive; between 5 and 6, inclusive; between 4 and5, inclusive; between 3 and 4, inclusive; between 2 and 3, inclusive; orbetween 1 and 2, inclusive. Similarly, a “basic linker” is a linker thathas a pI of greater than 7; between 7 and 8, inclusive; between 8 and 9,inclusive; between 9 and 10, inclusive; between 10 and 11, inclusive;between 11 and 12 inclusive, or between 12 and 13, inclusive. In certainembodiments, an acidic linker will contain a sequence that is selectedfrom the group consisting of [Gly-Glu]_(n) (SEQ ID NO:126);[Gly-Gly-Glu]_(n) (SEQ ID NO:127); [Gly-Gly-Gly-Glu]_(n) (SEQ IDNO:128); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO:129), [Gly-Asp]_(n) (SEQID NO:130); [Gly-Gly-Asp]_(n) (SEQ ID NO:131); [Gly-Gly-Gly-Asp]_(n)(SEQ ID NO:132); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO:133) where n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly-Glu]₆ (SEQ IDNO: 153). In certain embodiments, a basic linker will contain a sequencethat is selected from the group consisting of [Gly-Lys]_(n) (SEQ IDNO:134); [Gly-Gly-Lys]_(n) (SEQ ID NO:135); [Gly-Gly-Gly-Lys]_(n) (SEQID NO:136); [Gly-Gly-Gly-Gly-Lys]_(n) (SEQ ID NO:137), [Gly-Arg]_(n)(SEQ ID NO:138); [Gly-Gly-Arg]_(n) (SEQ ID NO:139);[Gly-Gly-Gly-Arg]_(n) (SEQ ID NO:140); [Gly-Gly-Gly-Gly-Arg]_(n) (SEQ IDNO:141) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example,[Gly-Gly-Lys]₆ (SEQ ID NO: 154).

Additionally, linkers may be prepared which possess certain structuralmotifs or characteristics, such as an a helix. For example, such alinker may contain an sequence that is selected from the groupconsisting of [Glu-Ala-Ala-Ala-Lys]_(n) (SEQ ID NO:142), where n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Glu-Ala-Ala-Ala-Lys]₃(SEQ ID NO: 155), [Glu-Ala-Ala-Ala-Lys]₄ (SEQ ID NO: 156), or[Glu-Ala-Ala-Ala-Lys]₅ (SEQ ID NO: 157).

Additionally, a non-peptidic linker may be employed to serve as the L1moiety of an engineered polypeptide described herein. For example, asunderstood in the art, an exemplary non-peptide linker such as a PEGlinker may be so-employed. See, e.g., WO2000024782. In certainembodiments, such a PEG linker has a molecular weight of 100 Da to 1000kDa. In certain embodiments, such a PEG linker has a molecular weight of100 Da to 500 kDa. In certain embodiments, such a PEG linker has amolecular weight of 100 Da to 100 kDa. In certain embodiments, such aPEG linker has a molecular weight of 100 Da to 50 kDa. In certainembodiments, such a PEG linker has a molecular weight of 100 Da to 10kDa. In certain embodiments, such a PEG linker has a molecular weight of100 Da to 5 kDa. In certain embodiments, such a PEG linker has amolecular weight of 100 Da to 1 kDa. In certain embodiments, such a PEGlinker has a molecular weight of 100 Da to 500 Da.

It is also to be understood that linkers suitable for use in accordancewith the invention may possess one or more of the characteristics andmotifs described above. For example, a linker may comprise an acidiclinker as well as a structural motif, such as an alpha helix. Similarly,a linker may comprise a basic linker and a structural motif, such as analpha helix. A linker may comprise an acidic linker, a basic linker, anda structural motif, such as an a helix. Additionally, it is also to beunderstood that engineered polypeptides in accordance with the inventionmay possess more than one linker, and each such linker may possess oneor more of the characteristics described above.

The linkers described herein are exemplary, and linkers within the scopeof this invention may be much longer and may include other residues. Inone embodiment, expressly excluded are engineered polypeptides in whichthe leptin compound is linked directly to the ABD without a linker.

In some embodiments, the engineered polypeptide includes an ABD at theN-terminal, and a HD1 at the C-terminal. Conversely, in someembodiments, the engineered polypeptide includes an ABD at theC-terminal, and a HD1 at the N-terminal. In some embodiments, either theN-terminal or the C-terminal is a leptin, a leptin fragment, or a leptinanalog. Preferably, the ABD is at the N-terminus of a leptin compound.Further to embodiments which include an ABD and a HD 1, the engineeredpolypeptide can have the structure ABD-HD 1 or HD1-ABD (both read in theN-terminal to C-terminal orientation).

It is understood that absent an express indication of the N-terminusand/or C-terminus of a engineered polypeptide set forth herein, theengineered polypeptide is to be read in the N-terminus to C-terminusorientation. For example, where HD1 is a leptin or analog thereof, theterms HD1-ABD, HD1-L1-ABD, HD1-ABD, and the like mean, in the absence ofan express indication of the N-terminus and/or the C-terminus, that theleptin compound resides at the N-terminus of the engineered polypeptide,and the ABD resides at the C-terminus. Conversely, if the N-terminusand/or C-terminus is expressly indicated, then the engineeredpolypeptide is to be read according to the express indication of theterminii. For example, the terms HD1_(C-term)-ABD, HD1-L1-ABD_(N-term)and the like mean that the ABD resides at the N-terminus of theengineered polypeptide, and HD1 resides at the C-terminus.

In some embodiments of the above described engineered polypeptides, HD1is human leptin or metreleptin. In some further embodiments, HD1 is aleptin analog as described herein. In some embodiments, the leptinanalog is leptin A100, A300 or A500.

In some embodiments, the engineered polypeptide described herein has anaffinity for serum albumin which is different than the affinity of theABD polypeptide alone, i.e., in the absence of a conjugated hormonedomain. In order to obtain effective association, the engineeredpolypeptide can have a binding affinity for serum albumin such that thedissociation constant K_(D) is, for example, less than about 10⁻⁶ M,10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³, 10⁻¹⁴ M oreven 10⁻¹⁵ M. In some embodiments, the affinity is not excessively tightsuch that the engineered polypeptide can dissociate from the albumin andelicit a biological response, for example binding to a receptor, forexample, a leptin receptor. The affinity can be measured as described inPCT Published Appl. No. WO 2009/016043, preferably to human serumalbumin, which is incorporated herein by reference in its entirety andfor all purposes, including without limitation assays and synthesismethods.

In some embodiments, an engineered polypeptide described herein issuperior to a corresponding compound having a different moiety that canextend plasma half-life (e.g., PEG or of Fc or albumin) conjugated witha hormone domain(s). In this context, the term “superior” refers to avariety of functional properties which could be weighed in theevaluation of a treatment for a disease or disorder. For example, theengineered polypeptide described herein could require less biologicallyactive (hormone domain) component, for example 1×, 2×, 3×, 4×, 5×, oreven less, than the corresponding compound having a different moietyconjugated with the hormone domain(s). For further example, theengineered polypeptide described herein could have higher potency, forexample, 1.5×, 2×, 3×, 4×, 5×, 10×, 20×, 50×, or even higher potency.

Engineered polypeptide compounds contemplated herein include thecompounds as set forth in Table 2 following.

TABLE 2 Selected exemplary engineered polypeptides Cmpd Sequence SEQ IDNO. 1 MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 594LKDAILAALPTGGGGASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC 2MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 595LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 3MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 596FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSASLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP 4MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 597FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGV EALKDAILAALP 5MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 598LKDAILAALPTGGGGSGGGSGGGSGGGSASISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLK NIEKQLDHIQGC 6MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 599LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 7MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 600FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEG VEALKDAILAALP 8MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 601LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 9MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 602LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 10MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 603LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 11MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 604LKDAILAALPTGLAEAAAKEAAAKEAAAKEAAAKEAAAKAAAASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEAS GYSTEVVALSRLQGSLQDMLQQLDLSPGC12 MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 605LKDAILAALPTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGS LQDMLQQLDLSPGC 13MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 606LKDAILAALPTGGKGGKGGKGGKGGKGGKASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGS LQDMLQQLDLSPGC 14MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 607LKDAILAALPGTGGGGASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC 15MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 608LKDAILAALPGTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGS LQDMLQQLDLSPGC 16MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 609FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSASLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEALKDAILAALPG 17MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 610FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGV EALKDAILAALPG 18MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 611LKDAILAALPGTGGGGSGGGSGGGSGGGSASISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSL KNIEKQLDHIQGC 19MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 612LKDAILAALPGTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQG SLQDMLWQLDLSPGC 20MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 613FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEG VEALKDAILAALPG 21MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 614LKDAILAALPGTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQ DMLRQLDRNPGC 22MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 615LKDAILAALPGTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQ DMLRQLDRNPGC 23MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 616LKDAILAALPGTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQG SLQDMLQQLDLSPGC 24MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 617LKDAILAALPGTGLAEAAAKEAAAKEAAAKEAAAKEAAAKAAAASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEA SGYSTEVVALSRLQGSLQDMLQQLDLSPGC25 MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 618LKDAILAALPGTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQ GSLQDMLQQLDLSPGC 26MGSLAEAKEAANAELDCYGVSDFYKRLIDKAKTVEGVEA 619LKDAILAALPGTGGKGGKGGKGGKGGKGGKASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQ GSLQDMLQQLDLSPGC 27MGSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 620LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 28MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 621FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGV EALKDAILAALP 29MGSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 622LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 30MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 623FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEG VEALKDAILAALP 31MGSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 624LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 32MGSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 625LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 33MGSLAQAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 626LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 34MGSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 627LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 35MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 628FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGV EALKDAILAALP 36MGSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 629LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 37MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 630FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEG VEALKDAILAALP 38MGSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 631LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 39MGSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 632LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 40MGSLAEAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 633LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 41MGSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 634LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 42MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 635FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGV EALKDAILAALP 43MGSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 636LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 44MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 637FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEG VEALKDAILAALP 45MGSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 638LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 46MGSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 639LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 47MGSLAQAKEAANRELDSYGVSDFYKRLIDKAKTVEGVEA 640LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 48MGSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEA 641LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 49MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 642FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGV EALKDAILAALP 50MGSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEA 643LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 51MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 644FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEG VEALKDAILAALP 52MGSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEA 645LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 53MGSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEA 646LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 54MGSLAEAKEAANRELDAYGVSDFYKRLIDKAKTVEGVEA 647LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 55MGSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEA 648LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 56MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 649FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGV EALKDAILAALP 57MGSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEA 650LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 58MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 651FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEG VEALKDAILAALP 59MGSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEA 652LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 60MGSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEA 653LKDAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQD MLRQLDRNPGC 61MGSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEA 654LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 62MGSLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEA 655LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 63MGSLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEA 656LKEAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 64MLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALK 657DAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDML RQLDRNPGC 65MLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALK 658DAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC 66MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 659LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQ DILQQLDLSPEC 67MGSLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEA 660LKDAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSL QDMLWQLDLSPGC 68MLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALK 661DAILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC 69MGSLAEAKVLANRELDKYGVSDFYKRLIEKAKTVEGVEA 662LKEAILAALPTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC 70MGSLAEAKVLANRELDKYGVSDFYKRLIDKAKTVEGVEA 663LKDAILAALPTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGS LQDMLQQLDLSPGC 71MLAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALK 681DAILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLR QLDRNPGC

Specifically contemplated are compounds of the above sequences in whichthe N-terminal methionine is absent, e.g. where the N-terminal commenceswith VPIQKV (SEQ ID NO: 158) or LAEAK (SEQ ID NO: 159) or GSLAEAK (SEQID NO: 35) for example, for leptin compounds. The N-terminal methionineis present primarily as a convenience for bacterial expression. However,conjugate peptides of the present invention can be expressed in aeukaryotic host cell (e.g. yeast (e.g. Pichia), mammalian, baculovirus)or other host cell having post-translational N-terminal proteolyticprocessing to yield an N-terminal amino acid as found in a naturallyoccurring mature peptide counterpart of the desired hormone or ABDsequence. Alternatively, an N-terminal sequence used for expressionand/or secretion can be one that can be removed post-translationally,e.g. as by use of a protease such as TEV.

III. Methods of Design and Production

Design of Constructs.

The engineered polypeptides described herein can be designed at theamino acid level. These sequences can then be back translated using avariety of software products known in the art such that the nucleotidesequence is optimized for the desired expression host, e.g. basedprotein expression, codon optimization, restriction site content. Forexample, the nucleotide sequence can be optimized for E. coli basedprotein expression and for restriction site content. Based on thenucleotide sequence of interest, overlapping oligonucleotides can beprovided for multistep PCR, as known in the art. These oligonucleotidescan be used in multiple PCR reactions under conditions well known in theart to build the cDNA encoding the protein of interest. For one exampleis 1× Amplitaq Buffer, 1.3 mM MgCl₂, 200 uM dNTPs, 4 U Amplitaq Gold,0.2 uM of each primer (AmpliTaq Gold, ABI), with cycling parameters: (94C:30 s, 58 C:1 min, 72 C: 1 min), 35 cycles.

Restriction sites can be added to the ends of the PCR products for usein vector ligation as known in the art. Specific sites can include Nde1and Xho1, such that the cDNA can then be in the proper reading frame ina pET45b expression vector (Novagen). By using these sites, anyN-terminal His Tag that are in this vector can be removed as thetranslation start site would then be downstream of the tag. Onceexpression constructs are completed, verification can be conduct bysequencing using e.g., T7 promoter primer, T7 terminator primer andstandard ABI BigDye Term v3.1 protocols as known in the art. Sequenceinformation can be obtained from e.g., an ABI 3730 DNA Analyzer and canbe analyzed using Vector NTI v.10 software (Invitrogen). Expressionconstructs can be designed in a modular manner such that linkersequences can be easily cut out and changed, as known in the art.

Protease recognition sites, known in the art or described herein, can beincorporated into constructs useful for the design, construction,manipulation and production of recombinant engineering polypeptidesdescribed herein.

General Methods of Production.

The engineered polypeptides described herein may be prepared usingbiological, chemical, and/or recombinant DNA techniques that are knownin the art. Exemplary methods are described herein and in U.S. Pat. No.6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO2009/011544; and US Publication No. 2007/0238669, the disclosures ofwhich are incorporated herein by reference in their entireties and forall purposes. Other methods for preparing the compounds are set forthherein.

The engineered polypeptides described herein may be prepared usingstandard solid-phase peptide synthesis techniques, such as an automatedor semiautomated peptide synthesizer. Briefly and generally, the ABD andtherapeutic hormonal peptide can be made separately and then conjugatedtogether or can be made as a single polypeptide. Thus the albuminbinding polypeptide, therapeutic hormone or engineered polypeptide mayalternatively be produced by non-biological peptide synthesis usingamino acids and/or amino acid derivatives having reactive side-chainsprotected, the non-biological peptide synthesis comprising step-wisecoupling of the amino acids and/or the amino acid derivatives to form apolypeptide according to the first aspect having reactive side-chainsprotected, removing the protecting groups from the reactive side-chainsof the polypeptide, and folding of the polypeptide in aqueous solution.Thus, normal amino acids (e.g. glycine, alanine, phenylalanine,isoleucine, leucine and valine) and pre-protected amino acid derivativesare used to sequentially build a polypeptide sequence, in solution or ona solid support in an organic solvent. When a complete polypeptidesequence is built, the protecting groups are removed and the polypeptideis allowed to fold in an aqueous solution. Each polypeptide according tothe present disclosure reversibly folds, with the ABD domain reversiblyfolding into a three helix bundle domain without added factors, andhence folds spontaneously. The engineered conjugate may be produced by amethod comprising producing an albumin binding polypeptide according toany method, e.g. as described herein, such as by non-biological peptidesynthesis, and conjugating the produced ABD polypeptide with thetherapeutic hormone defined herein. The ABDs herein folding completelyreversibly. This was assessed by circular dicroism spectra analysis; onespectrum taken at 20° C. and a second spectrum after heating to 90° C.followed by return to 20° C. During this procedure the Tm was determinedand found to be unchanged after the folding of the denaturedpolypeptide. Typically, using such techniques, an alpha-N-carbamoylprotected amino acid and an amino acid attached to the growing peptidechain on a resin are coupled at RT in an inert solvent (e.g.,dimethylformamide, N-methylpyrrolidinone, methylene chloride, and thelike) in the presence of coupling agents (e.g.,dicyclohexylcarbodiimide, 1-hydroxybenzo-triazole, and the like) in thepresence of a base (e.g., diisopropylethylamine, and the like). Thealpha-N-carbamoyl protecting group is removed from the resultingpeptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine,and the like) and the coupling reaction repeated with the next desiredN-protected amino acid to be added to the peptide chain. SuitableN-protecting groups are well known in the art, such ast-butyloxycarbonyl (tBoc) fluorenylmethoxycarbonyl (Fmoc), and the like.The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.).

For chemical synthesis solid phase peptide synthesis can be used for theengineered polypeptides, since in general solid phase synthesis is astraightforward approach with excellent scalability to commercial scale,and is generally compatible with relatively long engineeredpolypeptides. Solid phase peptide synthesis may be carried out with anautomatic peptide synthesizer (Model 430A, Applied Biosystems Inc.,Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc orFmoc chemistry (See Applied Biosystems User's Manual for the ABI 430APeptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70,Applied Biosystems, Inc., Foster City, Calif.) with capping.Boc-peptide-resins may be cleaved with HF (−5° C. to 0° C., 1 hour). Thepeptide may be extracted from the resin with alternating water andacetic acid, and the filtrates lyophilized. The Fmoc-peptide resins maybe cleaved according to standard methods (e.g., Introduction to CleavageTechniques, Applied Biosystems, Inc., 1990, pp. 6-12). Peptides may alsobe assembled using an Advanced Chem Tech Synthesizer (Model MPS 350,Louisville, Ky.).

The compounds described herein may also be prepared using recombinantDNA techniques using methods known in the art, such as Sambrook et al.,1989, MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold SpringHarbor. Non-peptide compounds may be prepared by art-known methods. Forexample, phosphate-containing amino acids and peptides containing suchamino acids, may be prepared using methods known in the art, such asdescribed in Bartlett et al, 1986, Biorg. Chem. 14:356-377.

The engineered polypeptides may alternatively be produced by recombinanttechniques well known in the art. See, e.g., Sambrook et al., 1989(Id.). These engineered polypeptides produced by recombinanttechnologies may be expressed from a polynucleotide. One skilled in theart will appreciate that the polynucleotides, including DNA and RNA,that encode such engineered polypeptides may be obtained from thewild-type cDNA, e.g. human leptin, taking into consideration thedegeneracy of codon usage, and may further engineered as desired toincorporate the indicated substitutions. These polynucleotide sequencesmay incorporate codons facilitating transcription and translation ofmRNA in microbial hosts. Such manufacturing sequences may readily beconstructed according to the methods well known in the art. See, e.g.,WO 83/04053, incorporated herein by reference in its entirety and forall purposes. The polynucleotides above may also optionally encode anN-terminal methionyl residue. Non-peptide compounds useful in thepresent invention may be prepared by art-known methods. For example,phosphate-containing amino acids and peptides containing such aminoacids may be prepared using methods known in the art. See, e.g.,Bartlett and Landen, 1986, Bioorg. Chem. 14: 356-77.

A variety of expression vector/host systems may be utilized to containand express a engineered polypeptide coding sequence. These include butare not limited to microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withbacterial expression vectors (e.g., Ti or pBR322 plasmid); or animalcell systems. Mammalian cells that are useful in recombinant proteinproductions include but are not limited to VERO cells, HeLa cells,Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.Exemplary protocols for the recombinant expression of the protein aredescribed herein and/or are known in the art.

As such, polynucleotide sequences are useful in generating new anduseful viral and plasmid DNA vectors, new and useful transformed andtransfected prokaryotic and eukaryotic host cells (including bacterial,yeast, and mammalian cells grown in culture), and new and useful methodsfor cultured growth of such host cells capable of expression of thepresent engineered polypeptides. The polynucleotide sequences encodingengineered polypeptides herein may be useful for gene therapy ininstances where underproduction of engineered polypeptides would bealleviated, or the need for increased levels of such would be met.

The present invention also provides for processes for recombinant DNAproduction of the present engineered polypeptides. Provided is a processfor producing the engineered polypeptides from a host cell containingnucleic acids encoding the engineered polypeptide comprising: (a)culturing the host cell containing polynucleotides encoding theengineered polypeptide under conditions facilitating the expression ofthe DNA molecule; and (b) obtaining the engineered polypeptide.

Host cells may be prokaryotic or eukaryotic and include bacteria,mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkeycells, baby hamster kidney cells, cancer cells or other cells), yeastcells, and insect cells.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important for correct insertion, folding and/orfunction. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, andthe like, have specific cellular machinery and characteristic mechanismsfor such post-translational activities, and may be chosen to ensure thecorrect modification and processing of the introduced foreign protein.

Alternatively, a yeast system may be employed to generate the engineeredpolypeptides of the present invention. The coding region of theengineered polypeptides DNA is amplified by PCR. A DNA encoding theyeast pre-pro-alpha leader sequence is amplified from yeast genomic DNAin a PCR reaction using one primer containing nucleotides 1-20 of thealpha mating factor gene and another primer complementary to nucleotides255-235 of this gene (Kurjan and Herskowitz, 1982, Cell, 30:933-43). Thepre-pro-alpha leader coding sequence and engineered polypeptide codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature engineered polypeptide. As taught by Rose andBroach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., SanDiego, Calif. (1990), the vector further includes an ADH2 transcriptionterminator downstream of the cloning site, the yeast “2-micron”replication origin, the yeast leu-2d gene, the yeast REP1 and REP2genes, the E. coli beta-lactamase gene, and an E. coli origin ofreplication. The beta-lactamase and leu-2d genes provide for selectionin bacteria and yeast, respectively. The leu-2d gene also facilitatesincreased copy number of the plasmid in yeast to induce higher levels ofexpression. The REP1 and REP2 genes encode proteins involved inregulation of the plasmid copy number.

The DNA construct described in the preceding paragraph is transformedinto yeast cells using a known method, e.g., lithium acetate treatment(Steams et al., 1990, Meth. Enz. 185: 280-297). The ADH2 promoter isinduced upon exhaustion of glucose in the growth media (Price et al.,1987, Gene 55:287). The pre-pro-alpha sequence effects secretion of thefusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature engineered polypeptides(Bitter et al., 1984, Proc. Natl. Acad. Sci. USA 81:5330-5334).

Engineered polypeptides of the invention may also be recombinantlyexpressed in yeast, e.g., Pichia, using a commercially availableexpression system, e.g., the Pichia Expression System (Invitrogen, SanDiego, Calif.), following the manufacturer's instructions. This systemalso relies on the pre-pro-alpha sequence to direct secretion, buttranscription of the insert is driven by the alcohol oxidase (AOX1)promoter upon induction by methanol. The secreted engineered polypeptideis purified from the yeast growth medium by, e.g., the methods used topurify said engineered polypeptide from bacterial and mammalian cellsupernatants.

Alternatively, the DNA encoding a engineered polypeptide may be clonedinto a baculovirus expression vector, e.g. pVL1393 (PharMingen, SanDiego, Calif.). This engineered-polypeptide-encoding vector is then usedaccording to the manufacturer's directions (PharMingen) or knowntechniques to infect Spodoptera frugiperda cells, grown for example insF9 protein-free media, and to produce recombinant protein. The proteinis purified and concentrated from the media using methods known in theart, e.g. a heparin-Sepharose column (Pharmacia, Piscataway, N.J.) andsequential molecular sizing columns (Amicon, Beverly, Mass.), andresuspended in appropriate solution, e.g. PBS. SDS-PAGE analysis can beused to characterize the protein, for example by showing a single bandthat confirms the size of the desired engineered polypeptide, as canfull amino acid amino acid sequence analysis, e.g. Edman sequencing on aProton 2090 Peptide Sequencer, or confirmation of its N-terminalsequence.

For example, the DNA sequence encoding the predicted mature engineeredpolypeptide may be cloned into a plasmid containing a desired promoterand, optionally, a leader sequence (see, e.g., Better et al., 1988,Science 240:1041-1043). The sequence of this construct may be confirmedby automated sequencing. The plasmid is then transformed into E. coli,strain MC1061, using standard procedures employing CaCl2 incubation andheat shock treatment of the bacteria (Sambrook et al., Id.). Thetransformed bacteria are grown in LB medium supplemented withcarbenicillin, and production of the expressed protein is induced bygrowth in a suitable medium. If present, the leader sequence will affectsecretion of the mature engineered polypeptide and be cleaved duringsecretion. The secreted recombinant engineered polypeptide is purifiedfrom the bacterial culture media by the method described herein.

Alternatively, the engineered polypeptides may be expressed in an insectsystem. Insect systems for protein expression are well known to those ofskill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The engineeredpolypeptide coding sequence is cloned into a nonessential region of thevirus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of a engineered polypeptidewill render the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which engineeredpolypeptide of the present invention is expressed (Smith et al., 1983,J. Virol. 46:584; Engelhard et al., 1994, Proc. Natl. Acad. Sci. USA91:3224-3227).

In another example, the DNA sequence encoding the engineeredpolypeptides may be amplified by PCR and cloned into an appropriatevector, for example, pGEX-3× (Pharmacia, Piscataway, N.J.). The pGEXvector is designed to produce a fusion protein comprisingglutathione-S-transferase (GST), encoded by the vector, and a proteinencoded by a DNA fragment inserted into the vector's cloning site. Theprimers for the PCR may be generated to include, for example, anappropriate cleavage site. The recombinant fusion protein may then becleaved from the GST portion of the fusion protein. ThepGEX-3×/engineered polypeptide construct is transformed into E. coliXL-1 Blue cells (Stratagene, La Jolla, Calif.), and individualtransformants are isolated and grown at 37° C. in LB medium(supplemented with carbenicillin) to an optical density at wavelength600 nm of 0.4, followed by further incubation for 4 hours in thepresence of 0.5 mM Isopropyl beta-D-thiogalactopyranoside (SigmaChemical Co., St. Louis, Mo.). Plasmid DNA from individual transformantsis purified and partially sequenced using an automated sequencer toconfirm the presence of the desired engineered polypeptide-encoding geneinsert in the proper orientation.

The fusion protein, when expected to be produced as an insolubleinclusion body in the bacteria, may be purified as described above or asfollows. Cells are harvested by centrifugation; washed in 0.15 M NaCl,10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/mL lysozyme (SigmaChemical Co.) for 15 min. at RT. The lysate is cleared by sonication,and cell debris is pelleted by centrifugation for 10 min. at 12,000×g.The fusion protein-containing pellet is resuspended in 50 mM Tris, pH 8,and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min.at 6000×g. The pellet is resuspended in standard phosphate bufferedsaline solution (PBS) free of Mg++ and Ca++. The fusion protein isfurther purified by fractionating the resuspended pellet in a denaturingSDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in0.4 M KCl to visualize the protein, which is excised and electroelutedin gel-running buffer lacking SDS. If the GST/engineered polypeptidefusion protein is produced in bacteria as a soluble protein, it may bepurified using the GST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe mature engineered polypeptide. The digestion reaction (20-40 μgfusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mLPBS) is incubated 16-48 hrs. at RT and loaded on a denaturing SDS-PAGEgel to fractionate the reaction products. The gel is soaked in 0.4 M KClto visualize the protein bands. The identity of the protein bandcorresponding to the expected molecular weight of the engineeredpolypeptide may be confirmed by partial amino acid sequence analysisusing an automated sequencer (Applied Biosystems Model 473A, FosterCity, Calif.).

In a particularly exemplary method of recombinant expression of theengineered polypeptides of the present invention, 293 cells may beco-transfected with plasmids containing the engineered polypeptides cDNAin the pCMV vector (5′ CMV promoter, 3′ HGH poly A sequence) and pSV2neo(containing the neo resistance gene) by the calcium phosphate method. Inone embodiment, the vectors should be linearized with ScaI prior totransfection. Similarly, an alternative construct using a similar pCMVvector with the neo gene incorporated can be used. Stable cell lines areselected from single cell clones by limiting dilution in growth mediacontaining 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days.Cell lines are screened for engineered polypeptides expression by ELISAor Western blot, and high-expressing cell lines are expanded for largescale growth.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside, G418; also, that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, beta-glucuronidase and its substrate, GUS, and luciferaseand its substrate, luciferin.

The engineered polypeptides of the present invention may be producedusing a combination of both automated peptide synthesis and recombinanttechniques. For example, either or both of: the leptin; a leptin analog,a active leptin fragment, or leptin derivative; and an ABD; andoptionally a linker; employed in the preparation of the engineeredpolypeptides as disclosed herein can be made synthetically orrecombinantly and then ligated together using methods known in the art,such as “native chemical ligation” and known variations thereof in whichan amide bond is formed joining the parent compounds. See for exampleU.S. Pat. No. 6,326,468, which is incorporated herein by reference foral purposes. Alternatively, for example, an engineered polypeptide ofthe present invention may contain a combination of modificationsincluding deletion, substitution, insertion and derivatization byPEGylation (or other moiety, e.g. polymer, fatty acyl chain, C-terminalamidation). Such an engineered polypeptide may be produced in stages. Inthe first stage, an intermediate engineered polypeptide containing themodifications of deletion, substitution, insertion, and any combinationthereof, may be produced by recombinant techniques as described. Thenafter an optional purification step as described herein, theintermediate engineered polypeptide is PEGylated (or subjected to otherchemical derivatization, e.g., acylation, C-terminal amidation) throughchemical modification with an appropriate PEGylating reagent (e.g., fromNeKtar Transforming Therapeutics, San Carlos, Calif.) to yield thedesired engineered polypeptide derivative. One skilled in the art willappreciate that the above-described procedure may be generalized toapply to a engineered polypeptide containing a combination ofmodifications selected from deletion, substitution, insertion,derivation, and other means of modification well known in the art andcontemplated by the present invention.

Peptides may be purified by any number of methods known in the art,including as described herein In one method peptides are purified byRP-HPLC (preparative and analytical) using a Waters Delta Prep 3000system. A C4, C8 or C18 preparative column (10μ, 2.2×25 cm; Vydac,Hesperia, Calif.) may be used to isolate peptides, and purity may bedetermined using a C4, C8 or C18 analytical column (5μ, 0.46×25 cm;Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) may bedelivered to the analytical column at a flow rate of 1.0 ml/min and tothe preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C., 20-24 h). Hydrolysates may be derivatized and analyzed by standardmethods (Cohen et al, THE PICO TAG METHOD: A MANUAL OF ADVANCEDTECHNIQUES FOR AMINO ACID ANALYSIS, pp. 11-52, Millipore Corporation,Milford, Mass. (1989)). Fast atom bombardment analysis may be carriedout by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may beperformed using cesium iodide or cesium iodide/glycerol. Plasmadesorption ionization analysis using time of flight detection may becarried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.

Engineered Polypeptide Expression Assay.

Methods are available for assaying the level of protein expression by ahost cell. Procedures useful for assaying the level of proteinexpression by a host cell are exemplified in the following typicalprotocol. About 25 μl BL21 E. coli cells are transformed with 2 ulplasmid DNA (expression vector for the engineered polynucleotide). Cellscan be plated and incubated overnight at 37 degrees C. or at roomtemperature (RT) over a 48-hr period. A single colony can be selectedand used to grow starter culture in 4 ml LB media with appropriateantibiotic for ˜6 hrs. Glycerol stocks can be prepared by adding 100 ul80% sterile glycerol to 900 ul stock, which can then be mixed gently andstored at −80 C. A 250 μl sample can be removed for TCP uninducedsample. An aliquot, for example, 2 ml of Magic media containingappropriate antibiotic can be inoculated with 5 μl starter culture,which can then be incubated overnight (up to 24 hrs) at 37 C, 300 rpm.As known in the art, Magic Media is autoinducing. Alternatively, 60 mlMagic Media containing appropriate antibiotic can be inoculated with 60μl starter culture in a 250 ml or 125 ml Thompson flask, which can thenbe incubated overnight (up to 24 hrs) at 30 C, 300 rpm. Afterincubation, 250 μl culture can be removed from each tube and the cellspelleted. The cell can be resuspended in 1 ml 50 mM Tris pH 8, 150 mMNaCl, to which can be added 0.1 volumes (100 ul) POP culture reagent and1 μl r-lysozyme (1:750 dilution in r-lysozyme buffer). The mixture canbe mixed well and incubated at least 10 min at RT. The preparation canthen be centrifuge 10 min at 14000×G. The supernatant (soluble fraction)can be removed and retained, and samples can be prepared for gelanalysis (15 μl+5 μl LDS). The remaining inclusion body pellet can beresuspended in 1 ml 1% SDS with sonication. The sample can be preparedfor gel analysis (15 ul+5 μl LDS). For uninduced samples, 1.0 volumesPOP culture reagent and 1 μl r-lysozyme (1:750 dilution in r-lysozymebuffer) can be added. The mixture can be mixed well and incubated atleast 10 min at RT. These samples may not need to be centrifuged. Thesample can then be prepared for gel analysis (15 μl+5 μl LDS). NU-PAGEgels (4-12%) non-reduced in 1×MES buffer can be run and stained withSimplyBlue microwave protocol. Destaining can be conducted overnight, asknown in the art. A gel image can be retained, and analyzed to determineprotein expression levels.

Inclusion Body Preparation.

For engineered polypeptides that are found in the inclusion bodyfraction, the following procedure can be beneficial. The cell pellet canbe resuspended in a minimum of 100 ml Lysis buffer for each 50 mlculture. Upon the addition of 30 ml, a 10 ml pipette can be used toresuspend, then the tube can be washed out with an additional 70 ml. Theresuspended cell solution can be multiply run, e.g., 4 passes, through amicrofluidizer at 100 PSI (min) taking care to keep chamber in ice waterthrough the entire process. The fluidized slurry can be centrifuged at14000×g, 20 min (e.g., JLA 10.5, 10,000 rpm, using 250 ml Nalgene®bottles). The inclusion body pellet can be resuspended on ice in chilledlysis buffer with stir bar and stir plate for 1 hour at 4 C afterdisruption with pipette tip. The pellet can be resuspended a second timein distilled H₂O with stir bar and stir plate for 1 hour at 4 C afterdisruption with pipette tip, followed by centrifugation at 14000×g, 15min. The supernatant can be removed and discarded. The resultant can bestored at −80 C.

Protein Purification.

As described herein, numerous methods are known for isolation ofexpressed polypeptides. The following is one example. Inclusion bodypellets can be solubilized in appropriate volume of solubilizationbuffer (8M urea or 8M guanidine, 50 mM Tris, 10 mM DTT, pH 7.75) for 1hour at RT. The solubilized pellets can be centrifuged for 20 min at 27000 g. Filtered (e.g., 0.4 um) supernatant can be transferred drop bydrop into appropriate volume of refolding buffer (50 mM Tris-HCl, 1 Murea, 0.8 M arginine, 4 mM cysteine, 1 mM cystamine; pH 8) at RT. Theresult can then be placed at 4° C. overnight or longer with gentlemixing. Samples can be concentrated and run on a gel filtration column(Superdex™ 75 26/60) at 1-2 ml/min in 4 C environment using a GEHealthsciences AKTAFPLC™. Appropriate protein containing fractions canbe identified via SDS-PAGE, pooled and run through a second gelfiltration column. Pooled protein can then be concentrated in Amiconfilter to appropriate concentration and assayed for endotoxin levelsusing, e.g., Endosafe® PTS Reader (Charles River), as known in the art.Once a protein sample has passed the endotoxin criteria, it can besterile filtered, dispensed into aliquots and run through qualitycontrol assays. Quality control assays can include analytical HPLC-SEC,non reducing SDS PAGE and RP HPLC-MS to obtain approximate mass.Proteins can be obtained in 1×PBS (137 mM sodium chloride, 2.7 mMpotassium chloride, 4.3 mM disodium phosphate, 1.4 mM monopotassiumphosphate, pH7.2), distributed into aliquots and flash frozen forstorage at −70 to −80° C.

IV. Methods of Use and Treating Disease

Indications.

A variety of diseases and disorders are contemplated to be beneficiallytreated by the polypeptide compounds and methods described herein.

Obesity and Overweight.

Obesity and its associated disorders including overweight are common andserious public health problems in the United States and throughout theworld. Upper body obesity is the strongest risk factor known for type 2diabetes mellitus and is a strong risk factor for cardiovasculardisease. Obesity is a recognized risk factor for hypertension,atherosclerosis, congestive heart failure, stroke, gallbladder disease,osteoarthritis, sleep apnea, reproductive disorders such as polycysticovarian syndrome, cancers of the breast, prostate, and colon, andincreased incidence of complications of general anesthesia. See, e.g.,Kopelman, 2000, Nature 404:635-43.

Obesity reduces life-span and carries a serious risk of theco-morbidities listed above, as well disorders such as infections,varicose veins, acanthosis nigricans, eczema, exercise intolerance,insulin resistance, hypertension hypercholesterolemia, cholelithiasis,orthopedic injury, and thromboembolic disease. See e.g., Rissanen et al,1990, Br. Med. J., 301:835-7. Obesity is also a risk factor for thegroup of conditions called insulin resistance syndrome, or “Syndrome X”and metabolic syndrome. The worldwide medical cost of obesity andassociated disorders is enormous.

The pathogenesis of obesity is believed to be multi-factoral. A problemis that, in obese subjects, nutrient availability and energy expendituredo not come into balance until there is excess adipose tissue. Thecentral nervous system (CNS) controls energy balance and coordinates avariety of behavioral, autonomic and endocrine activities appropriate tothe metabolic status of the animal. The mechanisms or systems thatcontrol these activities are broadly distributed across the forebrain(e.g., hypothalamus), hindbrain (e.g., brainstem), and spinal cord.Ultimately, metabolic (i.e., fuel availability) and cognitive (i.e.,learned preferences) information from these systems is integrated andthe decision to engage in appetitive (food seeking) and consummatory(ingestion) behaviors is either turned on (meal procurement andinitiation) or turned off (meal termination). The hypothalamus isthought to be principally responsible for integrating these signals andthen issuing commands to the brainstem. Brainstem nuclei that controlthe elements of the consummatory motor control system (e.g., musclesresponsible for chewing and swallowing). As such, these CNS nuclei haveliterally been referred to as constituting the “final common pathway”for ingestive behavior.

Neuroanatomical and pharmacological evidence support that signals ofenergy and nutritional homeostasis integrate in forebrain nuclei andthat the consummatory motor control system resides in brainstem nuclei,probably in regions surrounding the trigeminal motor nucleus. There areextensive reciprocal connection between the hypothalamus and brainstem.A variety of CNS-directed anti-obesity therapeutics (e.g., smallmolecules and peptides) focus predominantly upon forebrain substratesresiding in the hypothalamus and/or upon hindbrain substrates residingin the brainstem.

Obesity remains a poorly treatable, chronic, essentially intractablemetabolic disorder. Accordingly, a need exists for new therapies usefulin weight reduction and/or weight maintenance in a subject. Suchtherapies would lead to a profound beneficial effect on the subject'shealth. Methods and therapies employing the engineered peptidesdisclosed herein, either alone or in combination with other anti-obesityagents (see, e.g., WO 2009064298 and US 20080207512 may provide suchbeneficial effects.

Leptin Deficiency.

Leptin deficiency has been shown to result in obesity. One form ofleptin deficiency is congenital leptin deficiency, a rare geneticdisorder. See Montague et al., 1997, Nature 387: 903-908. Severe leptindeficiency can be a result of uncontrolled insulin-deficient diabetesmellitus that results from destruction of insulin-secreting β-cells. Itis theorized that the lack of insulin leads to synthesis and storage oftriglycerides in adipose tissue, which prevents weight gain and in turndramatically reduces plasma leptin levels since leptin is synthesized inadipose tissue. These and other Leptin deficiencies, and disease anddisorders that result from such deficiencies, can be treated with leptinreplacement therapy, such as via daily leptin or leptin agonistinjections. The engineered polypeptides described herein can provide amore convenient and advantageous therapeutic treatment of such diseasesand disorders.

Diabetes and Cardiovascular Disease.

Diabetes mellitus is recognized as a complex, chronic disease in which60% to 70% of all case fatalities among diabetic patients are a resultof cardiovascular complications. Diabetes is not only considered acoronary heart disease risk equivalent but is also identified as anindependent predictor of adverse events, including recurrent myocardialinfarction, congestive heart failure, and death following acardiovascular incident. The adoption of tighter glucose control andaggressive treatment for cardiovascular risk factors would be expectedto reduce the risk of coronary heart disease complications and improveoverall survival among diabetic patients. Yet, diabetic patients are twoto three times more likely to experience an acute myocardial infarctionthan non-diabetic patients, and diabetic patients live eight to thirteenyears less than non-diabetic patients.

Understanding the high risk nature of diabetic/acute myocardialinfarction patients, the American College of Cardiology/American HeartAssociation (“ACC/AHA”) clinical practice guidelines for the managementof hospitalized patients with unstable angina or non-ST-elevationmyocardial infarction (collectively referred to as “ACS”) recentlyrecognized that hospitalized diabetic patients are a special populationrequiring aggressive management of hyperglycemia. Specifically, theguidelines state that glucose-lowering therapy for hospitalizeddiabetic/ACS patients should be targeted to achieve preprandial glucoseless than 10 mg/dL, a maximum daily target than 180 mg/dL, and apost-discharge hemoglobin A1c less than 7%.

In a nationwide sample of elderly ACS patients, it was demonstrated thatan increase in 30-day mortality in diabetic patients corresponded withthe patients having higher glucose values upon admission to thehospital. See “Diabetic Coronary Artery Disease & Intervention,”Coronary Therapeutics 2002, Oak Brook, Ill., Sep. 20, 2002. There isincreasing evidence that sustained hyperglycemia rather than transientelevated glucose upon hospital admission is related to serious adverseevents. Although the ideal metric for hyperglycemia and vascular risk inpatients is not readily known, it appears that the mean glucose valueduring hospitalization is most predictive of mortality. In a separatestudy of ACS patients form over forty hospitals in the United States, itwas found that persistent hyperglycemia, as opposed to random glucosevalues upon admission to the hospital, was more predictive ofin-hospital mortality. See Acute Coronary Syndrome Summit: A State ofthe Art Approach, Kansas City, Mo., Sep. 21, 2002. Compared with glucosevalues upon admission, a logistic regression model of glucose controlover the entire hospitalization was most predictive of mortality. Therewas nearly a two-fold increased risk of mortality during hospitalizationfor each 10 mg/dL increase in glucose over 120 mg/dL. In a smallercohort of consecutive diabetic/ACS patients, there was a graded increasein mortality at one year with increasing glucose levels upon hospitaladmission. In the hospital setting, the ACC/AHA guidelines suggestinitiation of aggressive insulin therapy to achieve lower blood glucoseduring hospitalization.

It has been reported that leptin can have direct benefit to treatingdiabetes, particularly in type I diabetes and type II diabetes, with orwithout the presence of obesity, and more particularly in conditions oflow serum leptin. It has been reported that leptin replenishment reducedor prevented hyperinsulinemia, insulin resistance and hyperglycemia invarious animal models of diabetes type 1 and 2 with or without attendantobesity. For example, high leptin plasma levels generated either bypharmacological administration of leptin or with adenoviral gene therapyreduced hyperglycemia and associated increases of plasma glucagon levelsin STZ-induced diabetes, despite persistently low insulin levels.

Lipid Regulation Diseases.

As known in the art, lipodystrophy is characterized by abnormal ordegenerative conditions of the body's adipose tissue. Dyslipidemia is adisruption in the normal lipid component in the blood. It is believedthat prolonged elevation of insulin levels can lead to dyslipidemia.Hyperlipidemia is the presence of raised or abnormal levels of lipidsand/or lipoproteins in the blood. Hypothalamic amenorrhea is a conditionin which menstruation stops for several months due to a probleminvolving the hypothalamus. It has been found that leptin replacementtherapy in women with hypothalamic amenorrhea improves reproductive,thyroid, and growth hormone axes and markers of bone formation withoutcausing adverse effects. See e.g., Oral et al., N Engl J Med. 2004, 351:959-962, 987-997. Fatty liver disease, e.g., nonalcoholic fatty liverdisease (NAFLD) refers to a wide spectrum of liver disease ranging fromsimple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH),to cirrhosis (irreversible, advanced scarring of the liver). All of thestages of NAFLD have in common the accumulation of fat (fattyinfiltration) in the liver cells (hepatocytes). It is believed thatleptin is one of the key regulators for inflammation and progression offibrosis in various chronic liver diseases including NASH. See e.g.,Ikejima et al., Hepatology Res. 33:151-154.

Additionally, without wishing to be bound by any theory, it is believedthat relative insulin deficiency in type 2 diabetes, glucose toxicity,and increased hepatic free fatty acid burden through elevated deliveryfrom intra-abdominal adipose tissue via the portal vein, are implicatedas possible causes in fatty liver disorders. Indeed, it has beenhypothesized that eating behavior is the key factor driving themetabolic syndrome of obesity with its many corollaries, including NASH.Accordingly, treatments aimed at decreasing food intake and increasingthe number of small meals, as has already been demonstrated in type 2diabetes, may effectively treat and prevent NASH. Drugs that promoteinsulin secretion and weight loss, and delay gastric emptying are alsoeffective at improving glucose tolerance and thus may improve fattyliver with its attendant hyperinsulinemia. Thus, use of a leptin, leptinanalog, e.g., metreleptin, or an active fragment thereof, can be wellsuited as a treatment modality for this condition. Accordingly,engineered polypeptides described herein which include a leptin, leptinanalog or an active fragment thereof, can be useful in the treatment offatty liver disorders.

Alzheimer's Disease.

Alzheimer's disease (AD), as known in the art, is associated withplaques and tangles in the brain which include dysregulation of theA-beta protein. It is believed that brain lipids are intricatelyinvolved in A-beta-related pathogenic pathways, and that an importantmodulator of lipid homeostasis is leptin. Accordingly, leptin canmodulate bidirectional A-beta kinesis, reducing its levelsextracellularly. Indeed, it has been demonstrated that chronicadministration of leptin to AD-transgenic animals reduced the brainA-beta load, underlying its therapeutic potential. See Fewlass et al.,2004, FASEB J., 18:1870-1878. Additionally, type 2 diabetes mellitus andAD share epidemiological and biochemical features in that both arecharacterized by insoluble protein aggregates with a fibrillarconformation-amylin in type 2 DM pancreatic islets, and Aβ in AD brain.Without wishing to be bound by any theory, it is believed that similartoxic mechanisms may characterize type-2 DM and AD. See Lim et al., FEBSLett., 582:2188-2194.

Metabolic Syndrome X.

Metabolic Syndrome X is characterized by insulin resistance,dyslipidemia, hypertension, and visceral distribution of adipose tissue,and plays a pivotal role in the pathophysiology of type 2 diabetes. Ithas also been found to be strongly correlated with NASH, fibrosis, andcirrhosis of the liver. Accordingly, engineered polypeptides describedherein can be useful in the treatment of metabolic syndrome X.

Huntington's Disease.

Huntington's Disease is an autosomal dominant, neurogenerative disease.Features of the disease include motor disturbances, dementia,psychiatric problems, and unintended weight loss. Engineeredpolypeptides described herein can be useful in the treatment ofHuntington's Disease.

Accordingly, in one aspect, there is provided a method for treating adisease or disorder in a subject. The subject is in need of treatmentfor the disease or disorder. The disease or disorder can belipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity,hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fattyliver disease or diabetes (including type I and type II). Additionaldiseases and disorders which can be treated by the compounds and methodsdescribed herein include nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X andHuntington's Disease. The method of treatment includes administration tothe subject of an engineered polypeptide as described herein in anamount effective to treatment the disease or disorder. The engineeredpolypeptide will include as HD1 a leptin, a leptin fragment or a leptinanalog. Accordingly, the engineered polypeptide can have one of thefollowing structures: ABD-HD1, HD1-ABD, ABD-L1-HD1 or HD1-L1-ABD.

In all of the treatment embodiments described herein, the leptin can behuman leptin or metreleptin. In some embodiments, the leptin analog hasat least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or even higher, identity with human leptin. In someembodiments, the leptin analog has at least 50%, for example, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher, identitywith mouse leptin. In some embodiments, the leptin analog has at least50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or even higher, identity with rat leptin. In some embodiments, theleptin analog has at least 50%, for example, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or even higher, identity with platypusleptin. In some embodiments, the leptin analog has at least 50%, forexample, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or evenhigher, identity with seal leptin. In some embodiments, the leptinanalog is leptin A100, A300 or A500.

V. Assays

Methods for production and assay of engineered polypeptides describedherein are generally available to the skilled artisan. Further, specificmethods are described herein as well as in the patent publications andother references cited herein, which are incorporated by reference forthis additional purpose.

Food Intake.

Without wishing to be bound by any theory, it is believed that foodintake is useful in the assessment of the utility of a compound asdescribed herein. For example, it is known that a number of metabolicpathologies are related to food intake (e.g., diabetes, obesity).Accordingly, an initial screening can be conducted to determine theextent to which food intake is modulated by administration of compoundsdescribed herein, and a positive initial screening can be useful insubsequent development of a compound.

A variety of food intake assays are available to one of skill in theart. For example, in the so-called “home cage model” of food intake,subjects (e.g., rats) are maintained in their home cage, and food intakealong with total weight of the subject is measured following injectionof test compound. In the so-called “feeding patterns model” of foodintake assay, subjects (e.g., rats) are habituated to a feeding chamberand to injections prior to testing. After test compound administration,the subjects are immediately placed into the feeding chamber, and foodintake is automatically determined as a function of time (e.g., 1-minintervals). For both tests, the food is standard chow or any of avariety of chows (e.g., high fat) known in the art. In the so-called“mouse food intake” assay, a test compound may be tested for appetitesuppression, or for an effect on body weight gain in diet-inducedobesity (DIO) mice. In a typical mouse food intake assay, femaleNIH/Swiss mice (8-24 weeks old) are group housed with a 12:12 hourlight:dark cycle with lights on at 0600. Water and a standard pelletedmouse chow diet are available ad libitum, except as noted. Animals arefasted starting at approximately 1500 hrs, 1 day prior to experiment.The morning of the experiment, animals are divided into experimentalgroups. In a typical study, n=4 cages with 3 mice/cage. At time=0 min,all animals are given an intraperitoneal injection of vehicle orcompound, typically in an amount ranging from about 10 nmol/kg to 75nmol/kg, and immediately given a pre-weighed amount (10-15 g) of thestandard chow. Food is removed and weighed at various times, typically30, 60, and 120 minutes, to determine the amount of food consumed. Seee.g., Morley et al., 1994, Am. J. Physiol. 267:R178-R184). Food intakeis calculated by subtracting the weight of the food remaining at thee.g. 30, 60, 120, 180 and/or 240 minute time point, from the weight ofthe food provided initially at time=0. Significant treatment effects areidentified by ANOVA (p<0.05). Where a significant difference exists,test means are compared to the control mean using Dunnett's test (Prismv. 2.01, GraphPad Software Inc., San Diego, Calif.). For any testdescribed herein, administration of test compound can be by any means,including injection (e.g., subcutaneous, intraperitoneal, and the like),oral, or other methods of administration known in the art.

In Vitro Assays.

Without wishing to be bound by any theory or mechanism of action, it isbelieved that correlations exist between the results of in vitro (e.g.,receptor) assays, and the utility of agents for the treatment ofmetabolic diseases and disorders. Accordingly, in vitro assays (e.g.,cell based assays) are useful as a screening strategy for potentialmetabolic agents, such as described herein. A variety of in vitro assaysare known in the art, including those described as follows.

Leptin Binding Assay.

Leptin binding can be measured by the potency of a test compound indisplacing ¹²⁵I-recombinant-Leptin (murine) from the surface membraneexpressing chimeric Leptin (Hu)-EPO (Mu) receptor presented by the 32DOBECA cell line (J Biol Chem 1998; 273(29): 18365-18373). Purified cellmembranes can be prepared by homogenization from harvested confluentcell cultures of 32D OBECA cells. Membranes can be incubated with¹²⁵I-rec-Murine-Leptin and increasing concentrations of test compoundfor 3 hours at ambient temperature in 96-well polystyrene plates. Boundand unbound ligand fractions can then be separated by rapid filtrationonto 96-well GF/B plates pre-blocked for at least 60′ in 0.5% PEI(polyethyleneimine). Glass fiber plates can then be dried, scintillantadded, and CPM determined by reading on a multiwell scintillationcounter capable of reading radiolabeled iodine.

Leptin Functional Assay.

Increased levels of phosphorylated STAT5 (Signal Transducer andActivator of Transcription 5) can be measured following treatment of32D-Keptin cells ectopically expressing chimeric Hu-Leptin/Mu-EPOreceptor with a test compound. The 32D-Keptin cells (identical to32D-OBECA cells but maintained in culture with leptin) can be leptinweaned overnight and then treated with test compounds in 96-well platesfor 30 minutes at 37° C. followed by cell extraction. The pSTAT5 levelsin the cell lysates can be determined using the Perkin ElmerAlphaScreen® SureFire® pSTAT5 assay kit in a 384-well format(Proxiplate™ 384 Plus). The efficacy of test compounds can be determinedrelative to the maximal signal in cell lysates from cells treated withHuman leptin.

VI. Pharmaceutical Compositions

In one aspect, there are provided pharmaceutical compositions comprisingcompounds described herein in combination with a pharmaceuticallyacceptable excipient (e.g., carrier). The term “pharmaceuticallyacceptable carrier,” as used herein refers to pharmaceutical excipients,for example, pharmaceutically, physiologically, acceptable organic orinorganic carrier substances suitable for enteral or parenteralapplication that do not deleteriously react with the active agent.Suitable pharmaceutically acceptable carriers include water, saltsolutions (e.g., Ringer's solution and the like), alcohols, oils,gelatins, and carbohydrates such as lactose, amylose or starch, fattyacid esters, hydroxymethycellulose, and polyvinyl pyrrolidine. Suchpreparations can be sterilized and, if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,and/or aromatic substances and the like that do not deleteriously reactwith the compounds of the invention.

In a further aspect, there is provided a pharmaceutical compositionwhich includes a engineered polypeptide as described herein incombination with a pharmaceutically acceptable excipient. In someembodiments, the pharmaceutical composition is a long lastingpharmaceutical composition. The term “long lasting” in the context ofadministration of a pharmaceutical composition refers to duration ofaction. Accordingly, a long lasting pharmaceutical composition may beadministered at intervals of, for example, 1 hr, 2 hr, 4 hr, 8 hr, 12hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 1 month or even longer. In a preferred embodiment, administrationis once a day (i.e., “once daily”). In a more preferred embodiments,administration is once a week (i.e., “once weekly”).

A. Methods

The engineered polypeptides described herein can be administered aloneor can be co-administered to a subject. Co-administration is meant toinclude simultaneous or sequential administration of the compoundsindividually or in combination (more than one compound). For example, ithas been found that obesity can be beneficially treated with acombination therapy including a leptin (e.g., metreleptin) and certainother anti-obesity compounds. See e.g., U.S. Published Appl. No.2008/0207512. Accordingly, an engineered polypeptide described hereincomprising an ABD and a leptin could be useful for treatment of obesity.Alternatively, the individual engineered polypeptides having can beco-administered with other anti-obesity agents, such as exenatide orliraglutide.

In some embodiments, the formulations and methods described hereinfurther provide that the leptin or leptin analog engineered polypeptideis co-administered with one or more anti-diabetic agents, such asanti-hyperglycemia agents, e.g. insulin, amylins, pramlintide,metformin.

In some embodiments, the formulations and methods described hereinfurther provide that the leptin or leptin analog engineered polypeptideis co-administered with one or more cholesterol and/or triglyceridelowering agents. Exemplary agents include HMG CoA reductase inhibitors(e.g., atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin,simvastatin); bile ace sequestrants (e.g., colesevelam, cholestyramine,colestipol); fibrates (e.g., fenofibrate, clofibrate, gemfibrozil);ezetimibe, nicotinic acid, probucol, a lovastatin/niacin combination; anatorvastatin/amlodipine combination; and a simvastatin/ezetimibecombination.

The present disclosure provides the composition for use as a medicament,i.e. for use in therapy, since the leptin compound is a therapeuticallyactive compound, and surprisingly retains activity when fused to ABD.Compositions comprising an engineered polypeptide, either liquid or dryform, and optionally at least one pharmaceutically acceptable carrierand/or excipient are also specifically contemplated and are exemplifiedherein.

The composition has an ability to associate with albumin in vivo or invitro. In certain cases, it may be of benefit to form a complex of thecomposition with albumin outside of a living organism, i.e. to addexogenous albumin to the composition. Such a composition may belyophilized, providing a formulation that is suitable for storage atambient temperature. Thus, the present disclosure also provides acomposition as defined above which further comprises albumin, such ashuman serum albumin, and which may optionally be in dry form.

Co-administration can be achieved by separately administering theleptin, leptin analog, or leptin analog agonist engineered polypeptidewith the second agent, or by administering a single pharmaceuticalformulation comprising the leptin, leptin analog, or leptin analogagonist engineered polypeptide and the second agent. Appropriate dosageregimens for the second agents are generally known in the art.

The preparations can also be co-administered, when desired, with otheractive substances (e.g. to reduce metabolic degradation) as known in theart or other therapeutically active agents.

Amylins.

Amylin is a peptide hormone synthesized by pancreatic β-cells that isco-secreted with insulin in response to nutrient intake. The sequence ofamylin is highly preserved across mammalian species, with structuralsimilarities to calcitonin gene-related peptide (CGRP), the calcitonins,the intermedins, and adrenomedullin, as known in the art. Theglucoregulatory actions of amylin complement those of insulin byregulating the rate of glucose appearance in the circulation viasuppression of nutrient-stimulated glucagon secretion and slowinggastric emptying. In insulin-treated patients with diabetes,pramlintide, a synthetic and equipotent analogue of human amylin,reduces postprandial glucose excursions by suppressing inappropriatelyelevated postprandial glucagon secretion and slowing gastric emptying.The sequences of rat amylin, human amylin and pramlintide follow:

rat amylin: (SEQ ID NO: 108) KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY;human amylin: (SEQ ID NO: 109) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY;Pramlintide: (SEQ ID NO: 110) KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY.

Davalintide.

Davalintide, also known as “AC-2307” is a potent amylin agonist usefulin the treatment of a variety of disease indications. See WO 2006/083254and WO 2007/114838, each of which is incorporated by reference herein inits entirety and for all purposes. Davalintide is a chimeric peptide,having an N-terminal loop region of amylin or calcitonin and analogsthereof, an alpha-helical region of at least a portion of analpha-helical region of calcitonin or analogs thereof or analpha-helical region having a portion of an amylin alpha-helical regionand a calcitonin alpha-helical region or analog thereof, and aC-terminal tail region of amylin or calcitonin. The sequences of humancalcitonin, salmon calcitonin and davalintide follow:

human calcitonin: (SEQ ID NO: 111) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP;salmon calcitonin: (SEQ ID NO: 112) CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP;davalintide: (SEQ ID NO: 113) KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY.

Without wishing to be bound by any theory, it is believed that amylinsand davalintide, and fragments and analogs thereof, can requireC-terminal amidation to elicit a full biological response. It isunderstood that amylin compounds such as those described herein whichinclude amylins and/or davalintide, and fragment and analogs thereof,can be amidated at the C-terminal.

“Amylin agonist compounds” include native amylin peptides, amylin analogpeptides, and other compounds (e.g., small molecules) that have amylinagonist activity. The “amylin agonist compounds” can be derived fromnatural sources, can be synthetic, or can be derived from recombinantDNA techniques. Amylin agonist compounds have amylin agonist receptorbinding activity and may comprise amino acids (e.g., natural, unnatural,or a combination thereof), peptide mimetics, chemical moieties, and thelike. The skilled artisan will recognize amylin agonist compounds usingamylin receptor binding assays or by measuring amylin agonist activityin soleus muscle assays. In one embodiment, amylin agonist compoundswill have an IC₅₀ of about 200 nM or less, about 100 nM or less, orabout 50 nM or less, in an amylin receptor binding assay, such as thatdescribed herein, in U.S. Pat. No. 5,686,411, and US Publication No.2008/0176804, the disclosures of which are incorporated by referenceherein in their entireties and for all purposes. In one embodiment,amylin agonist compounds will have an EC₅₀ of about 20 nM or less, aboutnM 15 or less, about nM 10 or less, or about nM 5 or less in a soleusmuscle assay, such as that described herein and in U.S. Pat. No.5,686,411. In one embodiment, the amylin agonist compound has at least90% or 100% sequence identity to ^(25,28,29)Pro-human-amylin (SEQ ID NO:53). In one embodiment, the amylin agonist compound is a peptide chimeraof amylin (e.g., human amylin, rat amylin, and the like) and calcitonin(e.g., human calcitonin, salmon calcitonin, and the like). Suitable andexemplary amylin agonist compounds are also described in US PublicationNo. 2008/0274952, the disclosure of which is incorporated by referenceherein in its entirety and for all purposes.

By “amylin analog” as used herein is meant an amylin agonist that has atleast 50% sequence identity, preferably at least 70% sequence identity,to a naturally-occurring form of amylin, either rat or human or from anyother species, and is derived from them by modifications includinginsertions, substitutions, extensions, and/or deletions of the referenceamino acid sequence.

The amylin analog sequence can have at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 90%, or 95% amino acid sequence identity with the referenceamylin. In one aspect the analog has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or even 16 amino acid substitutions, insertions,extensions, and/or deletions relative to the reference compound. In oneembodiment, the amylin analog may comprise conservative ornon-conservative amino acid substitutions (including non-natural aminoacids and L and D forms). These analogs are preferably peptides, peptidederivatives or peptide mimics. Typical amylin analogs will be peptides,especially of 32-37 amino acids, e.g. 27 to 45, especially 28 to 38, andeven 31-36.

Amylin analogs with identity to rat and human amylin include^(25,28,29)Pro-h-amylin (pramlintide) (SEQ ID NO: 110);des-¹Lys-h-amylin (SEQ ID NO: 161); ²⁵Pro, ²⁶Val, ^(28,29)Pro-h-amylin(SEQ ID NO: 162); ¹⁸Arg, ^(25,28)Pro-h-amylin (SEQ ID NO: 163);des-¹Lys, ¹⁸Arg, ^(25,28)Pro-h amylin (SEQ ID NO: 164); ¹⁸Arg,^(25,28,29)Pro-h-amylin (SEQ ID NO: 165); des-¹Lys, ¹⁸Arg,^(25,28,29)Pro-h-amylin (SEQ ID NO: 166); des-¹,Lys^(25,28,29)Pro-h-amylin (SEQ ID NO: 167); ²⁵Pro, ²⁶Val,^(28,29)Pro-h-amylin (SEQ ID NO: 168); ²⁸Pro-h-amylin, 2,7-Cyclo-[²Asp,⁷Lys]-h-amylin (SEQ ID NO: 169); ²⁻³⁷h-amylin (SEQ ID NO: 170);¹Ala-h-amylin (SEQ ID NO: 171); ²Ala-h-amylin (SEQ ID NO: 172);^(2,7)Ala-h-amylin (SEQ ID NO: 173); ¹Ser-h-amylin (SEQ ID NO: 174);²⁹Pro-h-amylin (SEQ ID NO: 175); ^(25,28)Pro-h-amylin (SEQ ID NO: 176);des-¹Lys, ^(25,28)Pro-h-amylin (SEQ ID NO: 177); ²³Leu, ²⁵Pro, ²⁶Val,^(28,29)Pro-h-amylin (SEQ ID NO: 178); ²³Leu²⁵Pro²⁶Val²⁸Pro-h-amylin(SEQ ID NO: 179); des-¹Lys, ²³Leu, ²⁵Pro, ²⁶Val, ²⁸Pro-h-amylin (SEQ IDNO: 180); ¹⁸Arg, ²³Leu, ²⁵Pro, ²⁶Val, ²⁸Pro-h-amylin (SEQ ID NO: 181);¹⁸Arg, ²³Leu, ^(25,28,29)Pro-h-amylin (SEQ ID NO: 182); ¹⁸Arg²³Leu,^(25,28)Pro-h-amylin (SEQ ID NO: 183); ¹⁷Ile, ²³Leu,^(25,28,29)Pro-h-amylin (SEQ ID NO: 184); ¹⁷Ile, ^(25,28,29)Pro-h-amylin(SEQ ID NO: 185); des-¹Lys, ¹⁷Ile, ²³Leu, ^(25,28,29)Pro-h-amylin (SEQID NO: 186); ¹⁷Ile, ¹⁸Arg, ²³Leu-h-amylin (SEQ ID NO: 187); ¹⁷Ile,¹⁸Arg, ²³Leu, ²⁶Val, ²⁹Pro-h-amylin (SEQ ID NO: 188); ¹⁷Ile, ¹⁸Arg,²³Leu, ²⁵Pro, ²⁶Val, ^(28,29)Pro-h-amylin (SEQ ID NO: 189); ¹³Thr,²¹His, ²³Leu, ²⁶Ala, ²⁸Leu, ²⁹Pro, ³¹Asp-h-amylin (SEQ ID NO: 190);¹³Thr, ²¹His, ²³Leu, ²⁶Ala, ²⁹Pro, ³¹Asp-h-amylin (SEQ ID NO: 191);des-¹Lys, ¹³Thr, ²¹His, ²³Leu, ²⁶Ala, ²⁸Pro, ³¹Asp-h-amylin (SEQ ID NO:192); ¹³Thr, ¹⁸Arg, ²¹His, ²³Leu, ²⁶Ala, ²⁹Pro, ³¹Asp-h-amylin (SEQ IDNO: 193); ¹³Thr, ¹⁸Arg, ²¹His, ²³Leu, ^(28,29)Pro, ³¹Asp-h-amylin (SEQID NO: 194); and ¹³Thr, ¹⁸Arg, ²¹His, ²³Leu, ²⁵Pro, ²⁶Ala, ^(28,29)Pro,³¹Asp-h-amylin (SEQ ID NO: 195).

Suitable and exemplary amylin agonist compounds are also described inPCT Patent Publication WO2010/085700.

Amylin analogs include amino acid sequences of residues 1-37 of Formula(I) following, wherein up to 25% of the amino acids set forth in Formula(I) may be deleted or substituted with a different amino acid:

(SEQ ID NO: 800) X′-Xaa¹-Cys²-Asn³-Thr⁴-Ala⁵-Thr⁶-Cys⁷-Ala⁸-Thr⁹-Gln¹⁰-Arg¹¹-Leu¹²-Ala¹³-Asn¹⁴-phe¹⁵-Leu¹⁶-val¹⁷-His¹⁸-ser¹⁹-ser²⁰-Xaa²¹-Asn²²-Phe²³-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Thr³⁰-Xaa³¹-Val³²-Gly³³-Ser³⁴-Asn³⁵-Thr³⁶-Tyr³⁷-X (I).In Formula (I), X′ is hydrogen, an N-terminal capping group, or a linkerto a duration enhancing moiety. Xaa¹ is Lys or a bond, Xaa²¹ is Lys,Cys, or Asn, Xaa²⁴ is Lys, Cys, or Gly, Xaa²⁵ is Lys, Cys, or Pro, Xaa²⁶is Lys, Cys, or Ile, Xaa²⁷ is Lys, Cys, or Leu, Xaa²⁸ is Lys, Cys, orPro, Xaa²⁹ is Lys, Cys, or Pro and Xaa³¹ is Lys, Cys, or Asn. Furtherregarding Formula (I), the variable X represents a C-terminalfunctionality (e.g., a C-terminal cap). X is substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedcycloalkylamino, substituted or unsubstituted arylamino, substituted orunsubstituted aralkylamino, substituted or unsubstituted alkyloxy,substituted or unsubstituted aryloxy, substituted or unsubstitutedaralkyloxy, or hydroxyl. If the C-terminal of the polypeptide componentwith the sequence of residues 1-37 of Formula (I) is capped with afunctionality X, then X is preferably amine thereby forming a C-terminalamide. In some embodiments, up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45% or even 50% of the amino acids of residues 1-37 of Formula (I) aredeleted or substituted in a polypeptide component according to Formula(I). In some embodiments, the amylin analog component has 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or even 16 amino acidsubstitutions relative to the amino acid sequence set forth in Formula(I). In some embodiments, the amylin analog has a sequence which has adefined sequence identity with respect to the residues 1-37 of the aminoacid sequence according to Formula (I). In some embodiments, thesequence identity between an amylin analgo described herein and residues1-37 of Formula (I) is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or even higher. In some embodiments, up to 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5% or even less of the amino acids set forth in residues1-37 of Formula (I) may be deleted or substituted with a different aminoacid. In some embodiments, the sequence identity is within the range75%-100%. In some embodiments, the sequence identity is within the range75%-90%. In some embodiments, the sequence identity is within the range80%-90%. In some embodiments, the sequence identity is at least 75%. Insome embodiments, the amylin analog has the sequence of residues 1-37 ofFormula (I).

In some embodiments, amylin analogs including those of Formula (I), formthe basis of a polypeptide component to which one or more durationenhancing moieties are linked, optionally through a linker, to form anamylin polypeptide conjugate. Thus, the polypeptide component serves asa template (“polypeptide template”) to which is attached, preferably bycovalent attachment, one or more duration enhancing moieties. Linkage ofthe duration enhancing moiety to the polypeptide component can bethrough a linker as described herein. Alternatively, linkage of theduration enhancing moiety to the polypeptide component can be via adirect covalent bond. The duration enhancing moiety can be a watersoluble polymer as described herein. In some embodiments, a plurality ofduration enhancing moieties are attached to the polypeptide component,in which case each linker to each duration enhancing moiety isindependently selected from the linkers described herein.

Amylin analogs useful as polypeptide components described hereininclude, but are not limited to, the compounds set forth in residues1-37 of Formula (I) provided in Table 3 below. Unless indicated to thecontrary, all peptides described herein, including peptides having anexpressly provided sequence, are contemplated in both free carboxylateand amidated forms.

TABLE 3 Component polypeptides useful in the compounds described herein.Cmpd Description (sequence) 101KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO: 160) 102CNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO: 801)([desLys¹]-Cmpd 101) 103 KCNTATCATQRLANFLVRSSKNLGPVLPPTNVGSNTY-NH₂ (SEQID NO: 802) 104 CNTATCATQRLANFLVRSSKNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO:803) ([desLys¹]-Cmpd 103) 105 KCNTATCATQRLANFLVRSSNNLGPKLPPTNVGSNTY-NH₂(SEQ ID NO: 804) 106 CNTATCATQRLANFLVRSSNNLGPKLPPTNVGSNTY-NH₂ (SEQ IDNO: 805) ([desLys¹]-Cmpd 105) 107KCNTATCATQRLANFLVRSSNNLGPVLPPTKVGSNTY-NH₂ (SEQ ID NO: 806) 108CNTATCATQRLANFLVRSSNNLGPVLPPTKVGSNTY-NH₂ (SEQ ID NO: 807)([desLysi¹]-Cmpd 107) 109 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH₂ (SEQID NO: 808) 110 CNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH₂ (SEQ ID NO:809) ([desLys¹]-Cmpd 109) 111 CNTATCATQRLANFLVHSSKNFGPILPPTNVGSNTY-NH₂(SEQ ID NO: 810) 112 CNTATCATQRLANFLVHSSNNFGPKLPPTNVGSNTY-NH₂ (SEQ IDNO: 811) 113 CNTATCATQRLANFLVHSSNNFGPILPPTKVGSNTY-NH₂ (SEQ ID NO: 812)114 CNTATCATQRLANFLVHSSNNFKPILPPTNVGSNTY-NH₂ (SEQ ID NO: 813) 115CNTATCATQRLANFLVHSSNNFGKILPPTNVGSNTY-NH₂ (SEQ ID NO: 814) 116CNTATCATQRLANFLVHSSNNFGPIKPPTNVGSNTY-NH₂ (SEQ ID NO: 815) 117CNTATCATQRLANFLVHSSNNFGPILKPTNVGSNTY-NH₂ (SEQ ID NO: 816) 118CNTATCATQRLANFLVHSSNNFGPILPKTNVGSNTY-NH₂ (SEQ ID NO: 817)

The terms “linker” and the like, in the context of attachment ofduration enhancing moieties to a polypeptide component in an amylinpolypeptide conjugate described herein, means a divalent species (-L-)covalently bonded in turn to a polypeptide component having a valencyavailable for bonding and to a duration enhancing moiety having avalency available for bonding. The available bonding site on thepolypeptide component is conveniently a side chain residue (e.g.,lysine, cysteine, aspartic acid, and homologs thereof). In someembodiments, the available bonding site on the polypeptide component isthe side chain of a lysine or a cysteine residue. In some embodiments,the available bonding site on the polypeptide component is theN-terminal amine. In some embodiments, the available bonding site on thepolypeptide component is the C-terminal carboxyl. In some embodiments,the available bonding site on the polypeptide component is a backboneatom thereof. As used herein, the term “linking amino acid residue”means an amino acid within residues 1-37 of Formula (I) to which aduration enhancing moiety is attached, optionally through a linker.

In some embodiments, compounds are provided having a linker covalentlylinking a polypeptide component with a duration enhancing moiety. Thelinker is optional; i.e., any linker may simply be a bond. In someembodiments, the linker is attached at a side chain of the polypeptidecomponent. In some embodiments, the linker is attached to a backboneatom of the polypeptide component.

In another aspect, there is provided an amylin polypeptide conjugate,which is a derivative of pramlintide with SEQ ID NO:110 or an analogthereof, wherein the amino acid residue in position 1 is absent (i.e.,des-Lys¹) and an amino acid residue in position 2 to 37 has beensubstituted with a lysine residue or cysteine residue and wherein saidlysine residue or cysteine residue is linked to a polyethylene glycolpolymer, optionally via a linker, wherein the amino acid numberingconforms with the amino acid number in SEQ ID NO:110.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in any one ofposition 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 31, 32, 33, 34, 35, 36,or 37 is substituted with a lysine residue and wherein said lysineresidue is linked to a polyethylene glycol polymer, optionally via alinker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in any one ofposition 21, 24-29, or 31 is substituted with a lysine residue andwherein said lysine residue is linked to a polyethylene glycol polymer,optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 21 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 24 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 25 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 26 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 27 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 28 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 29 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys¹) and wherein an amino acid residue in position 31 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In some embodiments, the duration enhancing moiety is a water-solublepolymer. A “water soluble polymer” means a polymer which is sufficientlysoluble in water under physiologic conditions of e.g., temperature,ionic concentration and the like, as known in the art, to be useful forthe methods described herein. A water soluble polymer can increase thesolubility of a peptide or other biomolecule to which such water solublepolymer is attached. Indeed, such attachment has been proposed as ameans for improving the circulating life, water solubility and/orantigenicity of administered proteins, in vivo. See e.g., U.S. Pat. No.4,179,337; U.S. Published Appl. No. 2008/0032408. Many differentwater-soluble polymers and attachment chemistries have been used towardsthis goal, such as polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and the like.

In some embodiments, the linked duration enhancing moiety includes apolyethylene glycol. Polyethylene glycol (“PEG”) has been used inefforts to obtain therapeutically usable polypeptides. See e.g.,Zalipsky, S., 1995, Bioconjugate Chemistry, 6:150-165; Mehvar, R., 2000,J. Pharm. Pharmaceut. Sci., 3:125-136. As appreciated by one of skill inthe art, the PEG backbone [(CH₂CH₂—O—)_(n), n: number of repeatingmonomers] is flexible and amphiphilic. Without wishing to be bound byany theory or mechanism of action, the long, chain-like PEG molecule ormoiety is believed to be heavily hydrated and in rapid motion when in anaqueous medium. This rapid motion is believed to cause the PEG to sweepout a large volume and prevents the approach and interference of othermolecules. As a result, when attached to another chemical entity (suchas a peptide), PEG polymer chains can protect such chemical entity fromimmune response and other clearance mechanisms. As a result, pegylationcan lead to improved drug efficacy and safety by optimizingpharmacokinetics, increasing bioavailability, and decreasingimmunogenicity and dosing frequency. “Pegylation” refers in thecustomary sense to conjugation of a PEG moiety with another compound.For example, attachment of PEG has been shown to protect proteinsagainst proteolysis. See e.g., Blomhoff, H. K. et al., 1983, BiochimBiophys Acta, 757:202-208. Unless expressly indicated to the contrary,the terms “PEG,” “polyethylene glycol polymer” and the like refer topolyethylene glycol polymer and derivatives thereof, includingmethoxy-PEG (mPEG).

A variety of means have been used to attach polymer moieties such as PEGand related polymers to reactive groups found on the protein. See e.g.,U.S. Pat. No. 4,179,337; U.S. Pat. No. 4,002,531; Abuchowski et al.,1981, in “Enzymes as Drugs,” J. S. Holcerberg and J. Roberts, (Eds.),pp. 367-383; Zalipsky, S., 1995, Bioconjugate Chemistry, 6:150-165. Theuse of PEG and other polymers to modify proteins has been discussed. Seee.g., Cheng, T.-L. et al., 1999 m, Bioconjugate Chem., 10:520-528;Belcheva, N. et al., 1999, Bioconjugate Chem., 10:932-937; Bettinger, T.et al., 1998, Bioconjugate Chem., 9:842-846; Huang, S.-Y. et al., 1998,Bioconjugate Chem., 9:612-617; Xu, B. et al. 1998, Langmuir,13:2447-2456; Schwarz, J. B. et al., 1999, J. Amer. Chem. Soc.,121:2662-2673; Reuter, J. D. et al., 1999, Bioconjugate Chem.,10:271-278; Chan, T.-H. et al., 1997, J. Org. Chem., 62:3500-3504.Typical attachment sites in proteins include primary amino groups, suchas those on lysine residues or at the N-terminus, thiol groups, such asthose on cysteine side-chains, and carboxyl groups, such as those onglutamate or aspartate residues or at the C-terminus. Common sites ofattachment are to the sugar residues of glycoproteins, cysteines or tothe N-terminus and lysines of the target polypeptide. The terms“pegylated” and the like refer to covalent attachment of polyethyleneglycol to a polypeptide or other biomolecule, optionally through alinker as described herein and/or as known in the art.

In some embodiments, a PEG moiety in an amylin polypeptide conjugatedescribed herein has a nominal molecular weight within a specifiedrange. As customary in the art, the size of a PEG moiety is indicated byreference to the nominal molecular weight, typically provided inkilodaltons (kD). The molecular weight is calculated in a variety ofways known in the art, including number, weight, viscosity and “Z”average molecular weight. It is understood that polymers, such as PEGand the like, exist as a distribution of molecule weights about anominal average value.

Exemplary of the terminology for molecular weight for PEGs, the term“mPEG40KD” refers to a methoxy polyethylene glycol polymer having anominal molecular weight of 40 kilodaltons. Reference to PEGs of othermolecular weights follows this convention. In some embodiments, the PEGmoiety has a nominal molecular weight in the range 10-100 KD, 20-80 KD,20-60 KD, or 20-40 KD. In some embodiments, the PEG moiety has a nominalmolecular weight of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or even 100 KD. Preferably, the PEG moiety has amolecular weight of 20, 25, 30, 40, 60 or 80 KD.

PEG molecules useful for derivatization of polypeptides are typicallyclassified into linear, branched and Warwick (i.e., PolyPEG®) classes ofPEGs, as known in the art. Unless expressly indicated to the contrary,the PEG moieties described herein are linear PEGs. Furthermore, theterms “two arm branched,” “Y-shaped” and the like refer to branched PEGmoieties, as known in the art. The term “Warwick” in the context ofPEGs, also known as “comb” or “comb-type” PEGs, refers to a variety ofmulti-arm PEGs attached to a backbone, typically poly(methacrylate), asknown in the art. Regarding nomenclature including conventions employedin the table provided herein, absent indication to the contrary a PEGmoiety is attached to the backbone of the peptide. For example, Cmpd 119is the result of the conjugation of mPEG40KD to the N-terminal nitrogenof Cmpd 101. Similarly, Cmpd 120 is the result of conjugation ofmPEG40KD to the N-terminal nitrogen of Cmpd 102. Standard single letterabbreviations for amino acids can be used, as can standard three-letterabbreviations. For example, Cmpd 124 is an analog of Cmpd 110 whereinthe residue at position 26 of Cmpd 109 is substituted for lysine, andthe pendant amine functionality of lysine 26 (i.e., K²⁶) is conjugatedwith a PEG40KD moiety. Exemplary compounds are provided in Table 4below.

TABLE 4 Pegylated compounds Cmpd Description 119 mPEG40KD-Cmpd 101 (SEQID NO: 196) 120 mPEG40KD-Cmpd 102 (SEQ ID NO: 197) 121[K²¹(mPEG40KD)]-Cmpd 103 (SEQ ID NO: 198) 122 [K²¹(mPEG40KD)]-Cmpd 104(SEQ ID NO: 199) 123 [K²⁶(mPEG40KD)]-Cmpd 105 (SEQ ID NO: 200) 124[K²⁶(mPEG40KD)]-Cmpd 106 (SEQ ID NO: 201) 125 [K³¹(mPEG40KD)]-Cmpd 107(SEQ ID NO: 202) 126 [K³¹(mPEG40KD)]-Cmpd 108 (SEQ ID NO: 203) 127[K²⁶(Y-shaped-mPEG40KD)]-Cmpd 105 (SEQ ID NO: 204) 128[K²¹(mPEG40KD)]-Cmpd 111 (SEQ ID NO: 205) 129 [K²⁶(mPEG40KD)]-Cmpd 112(SEQ ID NO: 206) 130 [K³¹(mPEG40KD)]-Cmpd 113 (SEQ ID NO: 207) 131[K²⁶(Y-shaped-mPEG40KD)]-Cmpd 112 (SEQ ID NO: 208) 132[K²⁴(mPEG40KD)]-Cmpd 114 (SEQ ID NO: 209) 133 [K²⁵(mPEG40KD)]-Cmpd 115(SEQ ID NO: 210) 134 [K²⁷(mPEG40KD)]-Cmpd 116 (SEQ ID NO: 211) 135[K²⁸(mPEG40KD)]-Cmpd 117 (SEQ ID NO: 212) 136 [K²⁹(mPEG40KD)]-cmpd 118(SEQ ID NO: 213)

Amylins and amylin analogs to which a chemical moiety is attached areamylin derivatives. Amylin derivatives may constitute amylins to which achemical modification has been made of one or more of its amino acidside groups, α-carbon atoms, terminal amino group, or terminalcarboxylic acid group. A chemical modification includes, but is notlimited to, attaching one or more chemical moieties, creating new bonds,and removing one or more chemical moieties. Modifications at amino acidside groups include, without limitation, alkylation, acylation, esterformation, amide formation, maleimide coupling, acylation of lysineε-amino groups, N-alkylation of arginine, histidine, or lysine,alkylation of glutamic or aspartic carboxylic acid groups, anddeamidation of glutamine or asparagine. Modifications of the terminalamino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, and N-acyl modifications. Modifications of theterminal amino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, and N-acyl modifications, such as alkylacyls, branchedalkylacyls, alkylaryl-acyls. Modifications of the terminal carboxy groupinclude, without limitation, the amide, lower alkyl amide, dialkylamide, arylamide, alkylarylamide and lower alkyl ester modifications.Lower alkyl is C₁-C₄ alkyl. Furthermore, one or more side groups, orterminal groups, may be protected by protective groups known to theordinarily-skilled synthetic chemist. The α-carbon of an amino acid maybe mono- or dimethylated.

Amylin derivatives include amylins and amylin analogs conjugated to oneor more water soluble polymer molecules, such as polyethylene glycol(“PEG”), as described above, or fatty acid chains of various lengths(e.g., stearyl, palmitoyl, octanoyl), by the addition of polyaminoacids, such as poly-his, poly-arg, poly-lys, and poly-ala, or byaddition of small molecule substituents include short alkyls andconstrained alkyls (e.g., branched, cyclic, fused, adamantyl), andaromatic groups. In some embodiments, the water soluble polymermolecules will have a molecular weight ranging from about 500 Daltons toabout 60,000 Daltons. See, e.g., PCT Patent Publications WO 2007/104789,WO 2009/034119, and WO 2010/046357 for amylin derivatives suitable foruse as anti-obesity agents in combination with the engineeredpolypeptides of the invention.

Such polymer-conjugations may occur singularly at the N- or C-terminusor at the side chains of amino acid residues within the sequence of anamylin or amylin analog as disclosed herein. Alternatively, there may bemultiple sites of derivatization along the amino acid sequence of suchan amylin or amylin analog. Substitution of one or more amino acids withlysine, aspartic acid, glutamic acid, or cysteine may provide additionalsites for derivatization. In some embodiments, an amylin or amylinanalog may be conjugated to one, two, or three polymer molecules.

In some embodiments, the water soluble polymer molecules are linked toan amino, carboxyl, or thiol group, and may be linked by N or C termini,or at the side chains of lysine, aspartic acid, glutamic acid, orcysteine. Alternatively, the water soluble polymer molecules may belinked with diamine and dicarboxylic groups. In some embodiments, anamylin or amylin analog is conjugated to one, two, or three PEGmolecules through an epsilon amino group on a lysine amino acid.

It has been surprisingly discovered that the engineered polypeptides ofthe invention provide beneficial synergistic anti-obesity effects toboth moderately obese (BMI equal to or greater than 30) and severelyobese (BMI equal to or greater than 35) subjects when administered incombination with certain other anti-obesity compounds. As describedpreviously in, e.g., U.S. Published Appl. No. 2008/0207512, it has beenfound that a state of leptin resistance exists in obese subjects. Seealso, e.g., Tenenbaum, D., HHMI Bulletin, pp. 25-27 (March 2003);Chicurel, M., Nature, Vol. 404, pp. 538-540 (2000); Scarpace et al.,Diabetalogia, Vol. 48, pp. 1075-1083 (2005); and Bays et al., ObesityResearch, Vol. 12, (8), pp. 1197-1211 (2004). This leptin resistance,characterized at least in part by the presence of abnormally high serumleptin levels in obese subjects, makes these subjects unable to respondeffectively to leptin, whether endogenous or exogenously administered.It had been previously found that this leptin resistance could beovercome in moderately obese subjects, with a combination therapyincluding a leptin (e.g., metreleptin) and certain other anti-obesitycompounds. See e.g., U.S. Published Appl. No. 2008/0207512. It hasfurther been found that the synergistic anti-obesity effects of theleptin combination therapy are absent in severely obese, high BMIsubjects, presumably due to a severe leptin resistance. The inventorshave surprisingly discovered that the engineered compounds of theinvention are able to overcome even severe leptin resistance whenadministered in combination with certain other anti-obesity compounds.Accordingly, also provided by the invention are methods of treatingobesity and obesity related conditions, disorders, and diseases insubjects, including high BMI subjects, by the administration of at leasttwo different anti-obesity agents, wherein one anti-obesity agent is anengineered polypeptide of the invention and another anti-obesity agentis an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent).

In certain embodiments, the invention provides methods of treatingobesity in subjects in need thereof comprising administration of a firstanti-obesity agent selected from an engineered polypeptide of theinvention in combination with a second anti-obesity agent selected froman amylin, an amylin analog, an amylin agonist, or an amylin derivative,wherein the administration of the agents result in a synergistic effectas compared to administration of either agent alone.

In one aspect, methods of the invention provide a synergisticanti-obesity effect among the administered agents. Accordingly, incertain embodiments, administration of a combination of anti-obesityagents results in an effect, e.g., a reduction in nutrient availability,reduction in body weight, reduction in food intake, increase inmetabolism, which is greater than the combination of the results ofadministration of the anti-obesity agent alone (monotherapy).

“Reduced nutrient availability” is meant to include any means by whichthe body reduces the nutrients available to the body to store as fat. Inother words, reducing nutrient availability may be by means thatinclude, but are not limited to, reducing appetite, increasing satiety,affecting food choice/taste aversion, increasing metabolism, and/ordecreasing or inhibiting food absorption. Exemplary mechanisms that maybe affected include delayed gastric emptying or decreased absorption offood in the intestines.

As used herein, a “subject in need thereof” includes subjects who areoverweight, obese, or desirous of losing weight. Obese subjects includeboth the moderately obese, low BMI population and the severely obese,high BMI population. In addition, subjects who are insulin resistant,glucose intolerant, or have any form of diabetes mellitus (e.g., type 1,2 or gestational diabetes) can benefit from the methods of theinvention.

By “metabolic rate” is meant the amount of energy liberated/expended perunit of time. Metabolism per unit time can be estimated by foodconsumption, energy released as heat, or oxygen used in metabolicprocesses. It is generally desirable to have a higher metabolic ratewhen one wants to lose weight. For example, a person with a highmetabolic rate may be able to expend more energy (e.g., the body burnsmore calories) to perform an activity than a person with a low metabolicrate for that activity.

As used herein, “lean mass” or “lean body mass” refers to muscle andbone. Lean body mass does not necessarily indicate fat free mass. Leanbody mass contains a small percentage of fat (roughly 3%) within thecentral nervous system (brain and spinal cord), marrow of bones, andinternal organs. Lean body mass is measured in terms of density. Methodsof measuring fat mass and lean mass include, but are not limited to,underwater weighing, air displacement plethysmograph, x-ray, DEXA scans,MRIs and CT scans. In certain embodiments, fat mass and lean mass ismeasured using underwater weighing as known in the art.

By “fat distribution” is meant the location of fat deposits in the body.Such locations of fat deposition include, for example, subcutaneous,visceral and ectopic fat depots.

By “subcutaneous fat” is meant the deposit of lipids just below theskin's surface. The amount of subcutaneous fat in a subject can bemeasured using any method available for the measurement of subcutaneousfat. Methods of measuring subcutaneous fat are known in the art, forexample, those described in U.S. Pat. No. 6,530,886, the entirety ofwhich is incorporated herein by reference.

By “visceral fat” is meant the deposit of fat as intra-abdominal adiposetissue. Visceral fat surrounds vital organs and can be metabolized bythe liver to produce blood cholesterol. Visceral fat has been associatedwith increased risks of conditions such as polycystic ovary syndrome,metabolic syndrome and cardiovascular diseases.

By “ectopic fat storage” is meant lipid deposits within and aroundtissues and organs that constitute the lean body mass (e.g., skeletalmuscle, heart, liver, pancreas, kidneys, blood vessels). Generally,ectopic fat storage is an accumulation of lipids outside classicaladipose tissue depots in the body.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. “Treating” or “palliating” a disease, disorder, or conditionmeans that the extent and/or undesirable clinical manifestations of acondition, disorder, or a disease state are lessened and/or time courseof the progression is slowed or lengthened, as compared to not treatingthe disorder. For example, in treating obesity, a decrease in bodyweight, e.g., at least a 5% decrease in body weight, is an example of adesirable treatment result. For purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationor amelioration of one or more symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Further, treating does not necessarily occur by administration of onedose, but often occurs upon administration of a series of doses. Thus, atherapeutically effective amount, an amount sufficient to palliate, oran amount sufficient to treat a disease, disorder, or condition may beadministered in one or more administrations.

As used herein, the term “therapeutically effective amount” means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisorder being treated. The novel methods of treatment of this inventionare for disorders known to those skilled in the art.

As used herein, the term “prophylactically effective amount” means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician, to prevent the onset of obesity or an obesity-relateddisorder, condition or disease in subjects as risk for obesity or theobesity-related disorder, condition or disease.

In another aspect of the present invention, methods for reducing therisk of developing metabolic disorders are provided, where the methodcomprises administering to the subject a combination of anti-obesityagents in effective amounts to reduce the weight of a subject.

In some embodiments of the invention, methods of the invention are usedto increase the metabolic rate in a subject, decrease a reduction in themetabolic rate in a subject, or preserve the metabolic rate in asubject. In certain embodiments, the metabolic rate may involve thepreferential use of the body's fat as an energy source over lean bodytissue. In one aspect, lean body mass is not decreased followingadministration of the combination of anti-obesity agents. In anotheraspect, a reduction in the lean body mass is lessened or preventedfollowing administration of the combination of anti-obesity agents. Instill another aspect, lean body mass is increased followingadministration of the combination of anti-obesity agents. Suchpreference for fat as the energy source may be determined by comparingthe amount of fatty tissue to lean body tissue, ascertained by measuringtotal body weight and fat content at the beginning and end of thetreatment period. An increase in metabolic rate is a higher level of theuse of calories or another energy source by a subject over a period oftime compared with the level of use of calories or other energy sourceby the subject over another period of time under substantially similaror identical conditions without administration of the combination ofanti-obesity agents. In certain embodiments, the metabolic rate isincreased at least about 5% in a subject, in other embodiments, themetabolic rate is increased at least about 10%, 15%, 20% 25%, 30%, or35% in a subject compared with the level of use of calories or otherenergy source by the subject over another period of time undersubstantially similar or identical conditions without administration ofthe combination of anti-obesity agents. The increase in metabolic ratecan be measured using a respiratory calorimeter, for example. Aneffective amount of the anti-obesity agents as used in these embodimentsis an amount of each agent effective to increase the metabolic rate in asubject when administered in combination compared to a subject notreceiving the agents or only one of the agents.

In another embodiment, a method is provided to reduce a decrease inmetabolic rate in a subject. Such a decrease in metabolic rate can bethe result of any condition or nutritional or physical regimen thatleads to a reduction in metabolic rate, for example, due to a reducedcalorie diet, a restricted diet, or weight loss. A restricted dietincludes allowances or prohibitions, or both on the types of food or theamounts of food or both permitted in a diet, not necessarily based oncalories. For example, as in individual diets, the body compensates witha reduced metabolic rate based on the lower caloric intake. In essence,the body down-regulates the requirement for food, thereby subsisting onless food. As dieting continues, the threshold for caloric intake isreduced. When dieting has ended, the individual typically gains weightwhile eating a normal diet because of the lowered caloric intakethreshold and lower-basal metabolic rate (NIH Technology AssessmentConference Panel (1992) Ann. Intern. Med. 116:942-949; Wadden (1993)Ann. Intern. Med. 119:688-693). In one aspect, a method is provided toreduce the loss of metabolic rate in a subject, where the loss ofmetabolic rate is the result of a reduced calorie diet or weight loss.By using such a method, the subject's reduction in metabolic rate isdecreased by at least about 10%, 15%, 20% 25%, 30%, 35%, 40%, 50%, 60%,70%, 80%, 90%, or 95% in a subject. For such methods, it may bedesirable to administer the combination of anti-obesity agents at thetime the condition or nutritional or physical regimen is initiated whichleads to a loss or reduction in metabolic rate. However, it is alsocontemplated that administration of the agents is commenced before thecondition or nutritional or physical regimen is initiated. In oneinstance, metabolic rate is measured using a respiratory calorimeter. Aneffective amount of the anti-obesity agents of as used in thisembodiment is an amount of each agent effective to decrease thereduction of the metabolic rate in a subject when administered incombination.

In another aspect, methods for reducing metabolic plateaus are provided,where a method comprises administering effective amounts of anti-obesityagents in combination to a subject. In certain embodiments, the subjectis losing weight, or has lost weight, for example, due to a reducedcalorie diet, increased exercise or a combination thereof. By “metabolicplateau” is meant time intervals of steady metabolic rate while the bodyadjusts to changes in caloric or energy input. Changes in caloric inputor expenditure can be the result of, for example, reduced calorie dietsor increased physical activity. Such plateaus can be observed, forexample, during a weight loss regimen when weight loss slows or stops.In certain embodiments, a method of the present invention reduces theduration of a metabolic plateau in a subject compared with the durationof metabolic plateaus in an otherwise identical subject over the sameperiod of time under substantially similar or identical conditionswithout administration of the combination of anti-obesity agents. Inother embodiments, a method of the present invention reduces thefrequency of metabolic plateaus compared with the frequency of metabolicplateaus in an otherwise identical subject over the same period of timeunder substantially similar or identical conditions withoutadministration of the combination of anti-obesity agents. In still otherembodiments, a method of the present invention delays the onset of ametabolic plateau compared with the onset of a metabolic plateau in anotherwise identical subject over the same period of time undersubstantially similar or identical conditions without administration ofthe combination of anti-obesity agents. In certain embodiments,metabolic plateaus are identified by charting periods of reduced or noweight loss. In certain embodiments, at least one metabolic plateau isreduced. In other embodiments, at least two, three, four, five, six,seven, eight, nine, or ten metabolic plateaus are reduced. In anotheraspect, metabolic plateaus are delayed one day as compared to a subjectnot administered the combination of anti-obesity agents under identicalor similar conditions. In other aspects, metabolic plateaus are delayed2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks or 3weeks in a subject.

In yet other embodiments, a method is provided to preserve the metabolicrate in a subject. In certain embodiments, the subject may be at risk oflosing metabolic rate, for example, due to the initiation of a reducedcalorie diet, restricted diet, or anticipated weight loss. Apreservation of metabolic rate is a maintenance of the level of the useof calories or another energy source by a subject over a period of timecompared with the level of use of calories or other energy source by anotherwise identical subject over the same period of time undersubstantially similar or identical conditions without administration ofthe combination of anti-obesity agents. In one aspect, the metabolicrate is maintained within 15% of the subject's metabolic rate prior tothe initiation of the event that results in the decrease in metabolicrate. In other aspects, the metabolic rate is maintained within 10%,within 7%, within 5%, within 3% or less of the subject's metabolic rate.In one aspect, the combination of anti-obesity agents is administered atthe initiation of a reduced calorie diet, restricted diet, or exerciseregimen.

Metabolic rates can be assessed using any method available fordetermining such rates, for example by using a respiratory calorimeter.Such methods and devices for assaying metabolic rates are known in theart and are described, for example, in U.S. Pat. Nos. 4,572,208,4,856,531, 6,468,222, 6,616,615, 6,013,009, and 6,475,158.Alternatively, the metabolic rate of an animal can be assessed bymeasuring the amount of lean tissue versus fatty tissue catabolized bythe animal following the diet period. Thus, total body weight and fatcontent can be measured at the end of the dietary period. In rats, afrequently used method to determine total body fat is to surgicallyremove and weigh the retroperitoneal fat pad, a body of fat located inthe retroperitoneum, the area between the posterior abdominal wall andthe posterior parietal peritoneum. The pad weight is considered to bedirectly related to percent body fat of the animal. Since therelationship between body weight and body fat in rats is linear, obeseanimals have a correspondingly higher percent of body fat andretroperitoneal fat pad weight.

In another aspect of the present invention, methods for reducing fatmass by increasing the metabolic rate in a subject are provided, wherethe methods comprise administering a combination of anti-obesity agentsin amounts effective to reduce fat mass by increasing the subject'smetabolic rate. Fat mass can be expressed as a percentage of the totalbody mass. In some aspects, the fat mass is reduced by at least 1%, atleast 5%, at least 10%, at least 15%, at least 20%, or at least 25% overthe course of treatment. In one aspect, the subject's lean mass is notdecreased over the course of the treatment. In another aspect, thesubject's lean mass is maintained or increased over the course of thetreatment. In another aspect, the subject is on a reduced calorie dietor restricted diet. By “reduced calorie diet” is meant that the subjectis ingesting fewer calories per day than compared to the same subject'snormal diet. In one instance, the subject is consuming at least 50 fewercalories per day. In other instances, the subject is consuming at least100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 fewercalories per day.

In certain embodiments of the present invention, a method for alteringthe fat distribution in a subject is provided where the method comprisesadministering a combination of anti-obesity agents in amounts effectiveto alter fat distribution in the subject. In one aspect, the alterationresults from an increased metabolism of visceral or ectopic fat, or bothin the subject. In some embodiments, the method involves the metabolismof visceral or ectopic fat or both at a rate of at least about 5%, 10%,15%, 20%, 25%, 30%, 40%, or 50% greater than for subcutaneous fat. Inone aspect, the methods result in a favorable fat distribution. Incertain embodiments, favorable fat distribution is an increased ratio ofsubcutaneous fat to visceral fat, ectopic fat, or both. In one aspect,the method involves an increase in lean body mass, for example, as aresult of an increase in muscle cell mass.

In other embodiments, methods for reducing the amount of subcutaneousfat in a subject are provided, wherein the method comprisesadministering, to a subject in need thereof, a combination ofanti-obesity agents in amounts effective to reduce the amount ofsubcutaneous fat in the subject. In one instance, the amount ofsubcutaneous fat is reduced in a subject by at least about 5%. In otherinstances, the amount of subcutaneous fat is reduced by at least about10%, 15%, 20%, 25%, 30% 40%, or 50% compared to the subject prior toadministration of the anti-obesity agents.

The methods described herein can be used to reduce the amount ofvisceral fat in a subject. In one instance, the visceral fat is reducedin a subject by at least about 5%. In other instances, the visceral fatis reduced in the subject by at least about 10%, 15%, 20%, 25%, 30% 40%,or 50% compared to the subject prior to administration of thecombination of anti-obesity agents. Visceral fat can be measured throughany means available to determine the amount of visceral fat in asubject. Such methods include, for example, abdominal tomography bymeans of CT scanning and MRI. Other methods for determining visceral fatare described, for example, in U.S. Pat. Nos. 6,864,415, 6,850,797, and6,487,445.

In certain embodiments, a method for preventing the accumulation ofectopic fat or reducing the amount of ectopic fat in a subject isprovided, wherein the method comprises administering, to a subject inneed thereof, a combination of anti-obesity agents in amounts effectiveto prevent accumulation of ectopic fat or to reduce the amount ofectopic fat in the subject. In one instance, the amount of ectopic fatis reduced in a subject by at least about 5% compared to the subjectprior to administration of the combination of anti-obesity agents. Inother instances, the amount of ectopic fat is reduced in a subject by atleast about 10%, or by at least about 15%, 20%, 25%, 30% 40%, or 50%.Alternatively, the amount of ectopic fat is proportionally reduced 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% incomparison to subcutaneous fat in a subject. Ectopic fat can be measuredin a subject using any method available for measuring ectopic fat.

In other embodiments, methods are provided for producing a morefavorable fat distribution in a subject, where the method comprisesadministering to a subject a combination of anti-obesity agents inamounts effective to produce a favorable fat distribution. In certainembodiments, administration of a combination of anti-obesity agentsreduces the amount of visceral fat or ectopic fat, or both, in asubject. For example, administration of a combination of anti-obesityagents, where at least one anti-obesity agent that acts upon forebrainstructures involved in food intake or body weight modulation or both incombination with administration of at least one anti-obesity agent thatacts upon hindbrain structures involved in food intake or body weightmodulation or both. In certain embodiments, the methods preferentiallyreduce the amount of visceral or ectopic fat, or a combination of both,over the reduction in subcutaneous fat. Such methods result in a higherratio of subcutaneous fat to visceral fat or ectopic fat. Such improvedratios may result in a reduced risk of the development of cardiovasculardiseases, polycystic ovary syndrome, metabolic syndrome, or anycombinations thereof. In certain embodiments, ectopic or visceral fat ismetabolized at a rate 5% greater than subcutaneous fat. In otherembodiments, ectopic or visceral fat is metabolized at a rate at least10% 15%, 20%, 25%, 30% 50%, 60%, 70%, 80%, 90%, or 100% greater thansubcutaneous fat.

In still another aspect, methods of the invention include the use of atherapeutically effective amount of a combination of anti-obesity agentsadministered in combination with glucocortico steroids. Glucocorticosteroids have the adverse effect of increasing fat mass and decreasinglean mass. Accordingly, it is contemplated that the anti-obesity agentcombination can be used in conjunction with glucocortico steroids underconditions where glucocortico steroid use is beneficial.

In still another aspect, methods of the invention include the use of atherapeutically effective amount of one anti-obesity agent or acombination of anti-obesity agents administered in combination with atherapeutic agent selected from orlistat, phentermine, topiramate,CONTRAVE, and QNEXA. In some embodiments, the methods of the inventioninclude the use of a therapeutically effective amount of an engineeredpolypeptide of the invention in combination with a therapeutic agentselected from orlistat, phentermine, topiramate, CONTRAVE, and QNEXA. Inother embodiments, the methods of the invention include the use of atherapeutically effective amount of an amylin, an amylin analog, anamylin agonist, or an amylin derivative in combination with atherapeutic agent selected from orlistat, phentermine, topiramate,CONTRAVE, and QNEXA. In other embodiments, the methods of the inventioninclude the use of a therapeutically effective amount of an engineeredcompound of the invention in combination with an amylin, an amylinanalog, an amylin agonist, or an amylin derivative and a therapeuticagent selected from orlistat, phentermine, topiramate, CONTRAVE, andQNEXA.

Also provided are methods to reduce weight in a morbidly obese subjectby first reducing the subject's weight to a level below that of beingmorbidly obese, then administering to the subject a combination ofanti-obesity agents in effective amounts to further reduce the subject'sweight. Methods for reducing a subject's weight to below that of morbidobesity include reducing caloric intake, increasing physical activity,drug therapy, bariatric surgery, such as gastric bypass surgery, or anycombinations of the preceeding methods. In one aspect, administering thecombination of anti-obesity agents further reduces the weight of thesubject. In other embodiments, methods are provided for reducing thebody mass index in a subject having a body mass index of 40 or less byadministering a combination of anti-obesity agents in effective amountsto further reduce the subject's weight.

By reducing weight it is meant that the subject loses a portion ofhis/her total body weight over the course of treatment, whether thecourse of treatment be days, weeks, months or years. Alternatively,reducing weight can be defined as a decrease in proportion of fat massto lean mass (in other words, the subject has lost fat mass, butmaintained or gained lean mass, without necessarily a corresponding lossin total body weight). An effective amount of the anti-obesity agentsadministered in combination in these embodiments is an amount effectiveto reduce a subject's body weight over the course of the treatment, oralternatively an amount effective to reduce the subject's percentage offat mass over the course of the treatment. In certain embodiments, thesubject's body weight is reduced, over the course of treatment, by atleast about 1%, by at least about 5%, by at least about 10%, by at leastabout 15%, or by at least about 20%. Alternatively, the subject'spercentage of fat mass is reduced, over the course of treatment, by atleast 1%, at least 5%, at least 10%, at least 15%, at least 20%, or atleast 25%.

In certain embodiments, methods of reducing weight include improvedadherence to weight maintenance. Without wishing to be bound by anytheory, the restoration of leptin responsiveness achieved by theadministration of anti-obesity agents as described herein overcomes acritical challenge for obese subjects. In prior weight loss methods,leptin levels may still be higher than normal even at a reduced bodyweight, making it difficult for subjects to maintain the weight loss.The methods described herein include not only methods of reducingweight, but also the component of improved adherence to weightmaintenance.

In certain embodiments, methods of reducing nutrient availability, e.g.,reducing weight, in a subject comprise administering to the subject aneffective amount of the anti-obesity agents in a bolus dose one or moretimes a day. A bolus dose is an intermittent dosage of medicine (asopposed to a continuous infusion). A subject can be administered one ormore bolus doses per day. The bolus dose can be the same no matter whenit is administered to the subject, or can be adjusted such that thesubject is administered a larger bolus dose at certain times of the dayas compared to others. Administration of an agent in certainformulations, e.g., sustained-release formulations, a bolus dose can beadministered less frequently, for example, once every three days, onceper week, twice a month, once every month. Furthermore, the time betweenbolus doses is preferably long enough to allow the drug administered inthe previous bolus dose to clear the subject's blood stream.

In other embodiments, methods of reducing nutrient availability, e.g.,reducing weight, in a subject comprise administering to the subject aneffective amount of the anti-obesity agents in continuous doses. Bycontinuous dose it is intended to mean the continuous infusion of thedrug by, for example, intravenous injection or a transdermal patch.Alternatively, a continuous dose can be administered orally in the formof a controlled release capsule or tablet which releases the drug intothe subject's system over a period of time. When administered by acontinuous dose, the drug is released over a period of about 1 hour, insome cases the drug is released over a period of about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 18, or 24 hours.

By “administered in combination” is meant that the anti-obesity agentsare administered as a single administration, simultaneously as separatedoses, or as sequentially administered. Sequential administration refersto administering one of the anti-obesity agents either before or afteran anti-obesity agent. In certain embodiments, the first anti-obesityagent is administered about 30 minutes before or after the at least oneother anti-obesity agent, in other embodiments about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 hours before or after the at least one otheranti-obesity agents. Any of the administered anti-obesity agents can beadministered as a bolus dose or as a continuous dose.

Furthermore, in certain embodiments, administration of theweight-inducing agents in combination results in a synergistic effect inany of the aspects of the invention. In addition, in certainembodiments, administration of the weight-inducing agents in combinationresults in a lower dosage requirement for at least one of the agents,with the same effect.

Accordingly, in one embodiment is a method of treating obesity orreducing body weight in a subject in need thereof, comprisingperipherally administering therapeutically effective amounts of at leasttwo different anti-obesity agents, wherein at least one anti-obesityagent is an amylin, an amylin analog, an amylin agonist, or an amylinderivative and at least one anti-obesity agent is an engineeredpolypeptide comprising: an albumin binding domain polypeptide (ABD); anda first peptide hormone domain (HD1) selected from a leptin, a leptinanalog or an active fragment thereof, and the subject reduces bodyweight by least 10%, 12%, 15%, 20%, 30%, 40% or even 50%.

Further embodiments include the following.

Embodiment 1

A method of treating obesity in a subject comprising peripherallyadministering therapeutically effective amounts of at least twodifferent anti-obesity agents, wherein at least one anti-obesity agentis an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent) and at least one anti-obesity agent isan engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD); and a first peptide hormone domain (HD1) selectedfrom a leptin, a leptin analog or an active fragment thereof, whereinthe ABD comprises any one of the peptides selected from the groupconsisting of: SEQ ID NO:301-452, 455-463, and 500-593.

Embodiment 2

A method of reducing body weight in a subject comprising peripherallyadministering therapeutically effective amounts of at least twodifferent anti-obesity agents, wherein at least one anti-obesity agentis an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent) and at least one anti-obesity agent isan engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD); and a first peptide hormone domain (HD1) selectedfrom a leptin, a leptin analog or an active fragment thereof, whereinthe ABD comprises any one of the peptides selected from the groupconsisting of: SEQ ID NO:301-452, 455-463, and 500-593.

Embodiment 3

The method according to any one of embodiments 1 or 2 wherein the atleast one anti-obesity amylin agent is an amylin agonist.

Embodiment 4

The method according to any one of embodiments 1 to 3 wherein the amylinagonist comprises an amylin analog or derivative.

Embodiment 5

The method according to any one of embodiments 1 to 4 wherein the amylinanalog or derivative comprises pramlintide.

Embodiment 6

The method according to any one of embodiments 1 to 5 wherein the amylinanalog or derivative comprises a compound disclosed in Table 4.

Embodiment 7

The method according to any one of embodiments 1 to 6 wherein the amylinanalog or derivative comprises Des-Lys1-[Lys26(mPEG40K)]-Pramlintide(SEQ ID NO: 214).

Embodiment 8

The method according to any one of embodiments 1 to 7 wherein the HD1comprises an amino acid sequence selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO: 26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145,SEQ ID NO:146, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ IDNO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676,SEQ ID NO:677, and SEQ ID NO:680.

Embodiment 9

The method according to any one of embodiments 1 to 8, wherein the HD1is SEQ ID NO:29.

Embodiment 10

The method according to any one of embodiments 1 to 9, wherein theengineered polypeptide comprises a compound disclosed in Table 2.

Embodiment 11

The method according to any one of embodiments 1 to 10, wherein theengineered polypeptide comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO: 594 to 663 and 681.

Embodiment 12

The method according to any one of embodiments 1 to 11, wherein theengineered polypeptide comprises an amino acid sequence of SEQ IDNO:595.

Embodiment 13

The method according to any one of embodiments 1 to 11, wherein theengineered polypeptide comprises an amino acid sequence of SEQ IDNO:657.

Embodiment 14

The method according to any one of embodiments 1 to 13 wherein theeffective amount of the amylin agent and the effective amount of theengineered polypeptide comprises an amount such that a greater amount ofweight loss is achieved when the amylin agent is administered incombination with the engineered polypeptide to said subject than theamount of weight loss achieved when either agent is administered alone.

Embodiment 15

The method according to any one of embodiments 1 to 14 wherein the twoagents are administered at the same time.

Embodiment 16

The method according to any one of embodiments 1 to 15 wherein the twoagents are mixed together.

Embodiment 17

The method according to any one of embodiments 1 to 16 wherein thesubject's BMI is greater than 25.

Embodiment 18

The method according to any one of embodiments 1 to 17 wherein thesubject's BMI is 25 to 35.

Embodiment 19

The method according to any one of embodiments 1 to 18, wherein thesubject's BMI is 25 to 40.

Embodiment 20

The method according to any one of embodiments 1 to 19, wherein thesubject's BMI is 25 to 45.

Embodiment 21

The method according to any one of embodiments 1 to 20, wherein thesubject's BMI is 35 to 45.

Embodiment 22

The method according to any one of embodiments 1 to 21, wherein thesubject's BMI is reduced to less than 30.

Embodiment 23

The method according to any one of embodiments 1 to 22, wherein thesubject's BMI is reduced to less than 25.

Embodiment 24

The method according to any one of embodiments 1 to 23, wherein thesubject's BMI is reduced to normal.

Embodiment 25

The method according to any one of embodiments 1 to 24, wherein weightloss is achieved within 4 weeks of treatment.

Embodiment 26

The method according to any one of embodiments 1 to 25, wherein weightloss is achieved within 8 weeks of treatment.

Embodiment 27

The method according to any one of embodiments 1 to 26, wherein weightloss is achieved within 12 weeks of treatment.

Embodiment 28

The method according to any one of embodiments 1 to 27, wherein weightloss is achieved within 20 weeks of treatment.

Embodiment 29

The method according to any one of embodiments 1 to 28, wherein weightloss is achieved within 24 weeks of treatment.

Embodiment 30

The method according to any one of embodiments 1 to 29, wherein thesubject is human.

Embodiment 31

The method according to any one of embodiments 1 to 30, wherein thesubject is an obese human.

Embodiment 32

The method according to any one of embodiments 1 to 31, wherein theweight loss is reduced by at least 10%.

Embodiment 33

The method according to any one of embodiments 1 to 32, wherein theweight loss is reduced by at least 12%.

Embodiment 34

The method according to any one of embodiments 1 to 33, wherein theweight loss is reduced by at least 15%.

B. Formulations

The pharmaceutical compounds of the invention may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques such as those disclosed in Remington's PharmaceuticalSciences by E. W. Martin. See also Wang et al. (1988) J. of ParenteralSci. and Tech., Technical Report No. 10, Supp. 42:2 S.

In general, the engineered polypeptides may be formulated into a stable,safe pharmaceutical composition for administration to a patient.Pharmaceutical formulations contemplated for use in the methods of theinvention may comprise approximately 0.01 to 1.0% (w/v), in certaincases 0.05 to 1.0%, of the engineered polypeptide, approximately 0.02 to0.5% (w/v) of an acetate, phosphate, citrate or glutamate bufferallowing a pH of the final composition of from about 3.0 to about 7.0;approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcoholtonicifier and, optionally, approximately 0.005 to 1.0% (w/v) of apreservative selected from the group consisting of m-cresol, benzylalcohol, methyl, ethyl, propyl and butyl parabens and phenol. Such apreservative is generally included if the formulated peptide is to beincluded in a multiple use product.

In particular embodiments, a pharmaceutical formulation of the presentengineered polypeptides may contain a range of concentrations of thecompound(s), e.g., between about 0.01% to about 98% w/w, or betweenabout 1 to about 98% w/w, or preferably between 80% and 90% w/w, orpreferably between about 0.01% to about 50% w/w, or more preferablybetween about 10% to about 25% w/w in these embodiments. A sufficientamount of water for injection may be used to obtain the desiredconcentration of solution.

Additional tonicifying agents such as sodium chloride, as well as otherknown excipients, may also be present, if desired. In some cases, suchexcipients are useful in maintenance of the overall tonicity of thecompound. An excipient may be included in the presently describedformulations at various concentrations. For example, an excipient may beincluded in the concentration range from about 0.02% to about 20% w/w,preferably between about 0.02% and 0.5% w/w, about 0.02% to about 10%w/v, or about 1% to about 20% w/w. In addition, similar to the presentformulations themselves, an excipient may be included in solid(including powdered), liquid, semi-solid or gel form.

The pharmaceutical formulations may be composed in various forms, e.g.,solid, liquid, semisolid or liquid. The term “solid”, as used herein, ismeant to encompass all normal uses of this term including, for example,powders and lyophilized formulations. The presently describedformulations may be lyophilized.

The terms buffer, buffer solution and buffered solution, when used withreference to hydrogen-ion concentration or pH, refer to the ability of asystem, particularly an aqueous solution, to resist a change of pH onadding acid or alkali, or on dilution with a solvent. Characteristic ofbuffered solutions, which undergo small changes of pH on addition ofacid or base, is the presence either of a weak acid and a salt of theweak acid, or a weak base and a salt of the weak base. An example of theformer system is acetic acid and sodium acetate. The change of pH isslight as long as the amount of hydronium or hydroxyl ion added does notexceed the capacity of the buffer system to neutralize it.

As described herein, a variety of liquid vehicles are suitable for usein the formulations of engineered polypeptides, for example, water or anaqueous/organic solvent mixture or suspension.

The stability of a engineered polypeptide formulation for use asdescribed herein is enhanced by maintaining the pH of the formulation ina range determined by methods known in the art. In certain embodiments,the pH of the formulation is maintained in the range of about 3.5 to5.0, or about 3.5 to 6.5, in some embodiments from about 3.7 to 4.3, orabout 3.8 to 4.2. In some embodiments, pH may be about 4.0, about 5.0,about 6.0, about 7.0, about 8.0, about 9.0, or even higher. In someembodiments, pH may be in the physiological range, pH 6-8, preferably pH7-7.6.

In certain embodiments, the buffer with the engineered polypeptide is anacetate buffer (preferably at a final formulation concentration of fromabout 1-5 to about 60 mM), phosphate buffer (preferably at a finalformulation concentration of from about 1-5 to about to about 30 mM) orglutamate buffer (preferably at a final formulation concentration offrom about 1-5 to about to about 60 mM). In some embodiments, the bufferis acetate (preferably at a final formulation concentration of fromabout 5 to about 30 mM).

A stabilizer may be included in the formulations but is not necessarilyneeded. If included, however, a stabilizer useful in the practice of thepresent invention is a carbohydrate or a polyhydric alcohol. A suitablestabilizer useful in the practice of the present invention isapproximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol.The polyhydric alcohols and carbohydrates share the same feature intheir backbones, i.e., —CHOH—CHOH—, which is responsible for stabilizingthe proteins. The polyhydric alcohols include such compounds assorbitol, mannitol, glycerol, and polyethylene glycols (PEGs). Thesecompounds are straight-chain molecules. The carbohydrates, such asmannose, ribose, sucrose, fructose, trehalose, maltose, inositol, andlactose, on the other hand, are cyclic molecules that may contain a ketoor aldehyde group. These two classes of compounds have been demonstratedto be effective in stabilizing protein against denaturation caused byelevated temperature and by freeze-thaw or freeze-drying processes.Suitable carbohydrates include: galactose, arabinose, lactose or anyother carbohydrate which does not have an adverse affect on a diabeticpatient, i.e., the carbohydrate is not metabolized to form unacceptablylarge concentrations of glucose in the blood. Such carbohydrates arewell known in the art as suitable for diabetics. Sucrose and fructoseare suitable for use with the compound in non-diabetic applications(e.g. treating obesity).

In certain embodiments, if a stabilizer is included, the compound isstabilized with a polyhydric alcohol such as sorbitol, mannitol,inositol, glycerol, xylitol, and polypropylene/ethylene glycolcopolymer, as well as various polyethylene glycols (PEG) of molecularweight 200, 400, 1450, 3350, 4000, 6000, 8000 and even higher). Mannitolis the preferred polyhydric alcohol in some embodiments. Another usefulfeature of the lyophilized formulations of the present invention is themaintenance of the tonicity of the lyophilized formulations describedherein with the same formulation component that serves to maintain theirstability. In some embodiments, mannitol is the preferred polyhydricalcohol used for this purpose.

The United States Pharmacopeia (USP) states that anti-microbial agentsin bacteriostatic or fungistatic concentrations must be added topreparations contained in multiple dose containers. They must be presentin adequate concentration at the time of use to prevent themultiplication of microorganisms inadvertently introduced into thepreparation while withdrawing a portion of the contents with ahypodermic needle and syringe, or using other invasive means fordelivery, such as pen injectors. Antimicrobial agents should beevaluated to ensure compatibility with all other components of theformula, and their activity should be evaluated in the total formula toensure that a particular agent that is effective in one formulation isnot ineffective in another. It is not uncommon to find that a particularantimicrobial agent will be effective in one formulation but noteffective in another formulation.

A preservative is, in the common pharmaceutical sense, a substance thatprevents or inhibits microbial growth and may be added to pharmaceuticalformulations for this purpose to avoid consequent spoilage of theformulation by microorganisms. While the amount of the preservative isnot great, it may nevertheless affect the overall stability of thepeptide.

While the preservative for use in the pharmaceutical compositions canrange from 0.005 to 1.0% (w/v), in some embodiments range for eachpreservative, alone or in combination with others, is: benzyl alcohol(0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combinationof methyl (0.05-0.25%) and ethyl or propyl or butyl (0.005%-0.03%)parabens. The parabens are lower alkyl esters of para-hydroxybenzoicacid. A detailed description of each preservative is set forth inRemington's Pharmaceutical Sciences (Id.)

Engineered polypeptides may not have a tendency to adsorb onto the glassin a glass container when in a liquid form, therefore, a surfactant maynot be required to further stabilize the pharmaceutical formulation.However, with regard to compounds which do have such a tendency when inliquid form, a surfactant should be used in their formulation. Theseformulations may then be lyophilized. Surfactants frequently causedenaturation of protein, both of hydrophobic disruption and by saltbridge separation. Relatively low concentrations of surfactant may exerta potent denaturing activity, because of the strong interactions betweensurfactant moieties and the reactive sites on proteins. However,judicious use of this interaction can stabilize proteins againstinterfacial or surface denaturation. Surfactants which could furtherstabilize the engineered polypeptide may optionally be present in therange of about 0.001 to 0.3% (w/v) of the total formulation and includepolysorbate 80 (i.e., polyoxyethylene (20) sorbitan monooleate), CHAPS®(i.e., 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate), Brij®(e.g., Brij® 35, which is (polyoxyethylene (23) lauryl ether),poloxamer, or another non-ionic surfactant.

It may also be desirable to add sodium chloride or other salt to adjustthe tonicity of the pharmaceutical formulation, depending on thetonicifier selected. However, this is optional and depends on theparticular formulation selected. Parenteral formulations preferably maybe isotonic or substantially isotonic.

A preferred vehicle for parenteral products is water. Water of suitablequality for parenteral administration can be prepared either bydistillation or by reverse osmosis. Water for injection is the preferredaqueous vehicle for use in the pharmaceutical formulations.

It is possible that other ingredients may be present in thepharmaceutical formulations. Such additional ingredients may include,e.g., wetting agents, emulsifiers, oils, antioxidants, bulking agents,tonicity modifiers, chelating agents, metal ions, oleaginous vehicles,proteins (e.g., human serum albumin, gelatin or proteins) and azwitterion (e.g., an amino acid such as betaine, taurine, arginine,glycine, lysine and histidine). Additionally, polymer solutions, ormixtures with polymers provide the opportunity for controlled release ofthe peptide. Such additional ingredients, of course, should notadversely affect the overall stability of the pharmaceutical formulationof the present invention.

Containers are also an integral part of the formulation of an injectionand may be considered a component, for there is no container that istotally inert, or does not in some way affect the liquid it contains,particularly if the liquid is aqueous. Therefore, the selection of acontainer for a particular injection must be based on a consideration ofthe composition of the container, as well as of the solution, and thetreatment to which it will be subjected. Adsorption of the peptide tothe glass surface of the vial can also be minimized, if necessary, byuse of borosilicate glass, for example, Wheaton Type I borosilicateglass #33 (Wheaton Type I-33) or its equivalent (Wheaton Glass Co.).Other vendors of similar borosilicate glass vials and cartridgesacceptable for manufacture include Kimbel Glass Co., West Co., BunderGlas GMBH and Form a Vitrum. The biological and chemical properties ofthe compound may be stabilized by formulation and lyophilization in aWheaton Type I-33 borosilicate serum vial to a final concentration of0.1 mg/ml and 10 mg/ml of the compound in the presence of 5% mannitol,and 0.02% Tween 80.

For formulations to be delivered by injection, in order to permitintroduction of a needle from a hypodermic syringe into a multiple-dosevial and provide for resealing as soon as the needle is withdrawn, theopen end of each vial is preferably sealed with a rubber stopper closureheld in place by an aluminum band.

Stoppers for glass vials, such as, West 4416/50, 4416/50 (Teflon faced)and 4406/40, Abbott 5139 or any equivalent stopper can be used as theclosure for pharmaceutical for injection. For formulations comprisingpeptidic anti-obesity agents, these stoppers are compatible with thepeptide as well as the other components of the formulation. Theinventors have also discovered that these stoppers pass the stopperintegrity test when tested using patient use patterns, e.g., the stoppercan withstand at least about 100 injections. Alternatively, the peptidecan be lyophilized in to vials, syringes or cartridges for subsequentreconstitution. Liquid formulations of the present invention can befilled into one or two chambered cartridges, or one or two chambersyringes.

Each of the components of the pharmaceutical formulation described aboveis known in the art and is described in PHARMACEUTICAL DOSAGE FORMS:PARENTERAL MEDICATIONS, Vol. 1, 2nd ed., Avis et al. Ed., Mercel Dekker,New York, N.Y. 1992, which is incorporated by reference in its entiretyherein and for all purposes.

The manufacturing process for the above liquid formulations generallyinvolves compounding, sterile filtration and filling steps. Thecompounding procedure involves dissolution of ingredients in a specificorder (preservative followed by stabilizer/tonicity agents, buffers andpeptide) or dissolving at the same time.

Alternative formulations, e.g., non-parenteral, may not requiresterilization. However, if sterilization is desired or necessary, anysuitable sterilization process can be used in developing the peptidepharmaceutical formulation of the present invention. Typicalsterilization processes include filtration, steam (moist heat), dryheat, gases (e.g., ethylene oxide, formaldehyde, chlorine dioxide,propylene oxide, beta-propiolacctone, ozone, chloropicrin, peraceticacid methyl bromide and the like), exposure to a radiation source, andaseptic handling. Filtration is the preferred method of sterilizationfor liquid formulations of the present invention. The sterile filtrationinvolves filtration through 0.45 um and 0.22 um (1 or 2) which may beconnected in series. After filtration, the solution is filled intoappropriate vials or containers.

In certain embodiments, the engineered polypeptides described herein areadministered peripherally to the subjects. In some embodiments, theliquid pharmaceutical formulations of the present invention are intendedfor parenteral administration. Suitable routes of administration includeintramuscular, intravenous, subcutaneous, intradermal, intraarticular,intrathecal and the like. In some embodiments, the subcutaneous route ofadministration is preferred. In certain embodiments, mucosal delivery isalso preferred. These routes include, but are not limited to, oral,nasal, sublingual, pulmonary and buccal routes which may includeadministration of the peptide in liquid, semi-solid or solid form. Forformulations comprising engineered polypeptides, administration viathese routes can require substantially more compound to obtain thedesired biological effects due to decreased bioavailability compared toparenteral delivery. In addition, parenteral controlled release deliverycan be achieved by forming polymeric microcapsules, matrices, solutions,implants and devices and administering them parenterally or by surgicalmeans. Examples of controlled release formulations are described in U.S.Pat. Nos. 6,368,630, 6,379,704, and 5,766,627, which are incorporatedherein by reference. These dosage forms may have a lower bioavailabilitydue to entrapment of some of the peptide in the polymer matrix ordevice. See e.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and 6,296,842,each of which is incorporated herein by reference in its entirety andfor all purposes.

The compounds may be provided in dosage unit form containing an amountof the engineered polypeptide that will be effective in one or multipledoses.

As will be recognized by those in the field, an effective amount of theengineered polypeptide will vary with many factors including the age andweight of the subject, the subject's physical condition, the conditionto be treated, and other factors known in the art. An effective amountof the engineered polypeptides will also vary with the particularcombination administered. As described herein, administration of theengineered polypeptides in combination may allow for a reduced amount ofany of the administered engineered polypeptides to be an effectiveamount.

The long-duration of action of the engineered polypeptide can providethe extended duration of action desired, such as once daily or onceweekly administration. The duration of action can be selected, forexample, by choice of ABD and its affinity for albumin. While notwishing to be bound by theory, it is believed that higher affinity toalbumin will yield longer circulation times providing longer duration ofaction. Either or both pharmacodynamic (therapeutic effects) andpharmacokinetic (drug properties) can be measured over time afterdelivery, such as drug plasma levels, acute or chronic glucose and/orHbA1c lowering, insulin plasma levels, food intake inhibition or weightloss.

C. Effective Dosages

Pharmaceutical compositions provided herein include compositions whereinthe active ingredient is contained in a therapeutically effectiveamount, i.e., in an amount effective to achieve its intended purpose.The actual amount effective for a particular application will depend,inter alia, on the condition being treated. For example, whenadministered in methods to treat diabetes, such compositions willcontain an amount of active ingredient effective to achieve the desiredresult (e.g. decreasing fasting blood glucose in a subject). Whenadministered in methods to treat obesity, such compositions will containan amount of active ingredient effective to achieve the desired result(e.g. decrease the body mass).

The dosage and frequency (single or multiple doses) of compoundadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated (e.g., the disease responsive to compoundsdescribed herein); presence of other diseases or other health-relatedproblems; kind of concurrent treatment; and complications from anydisease or treatment regimen. Other therapeutic regimens or agents canbe used in conjunction with the methods and compounds of the invention.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring one or morephysiological parameters, including but not limited to blood sugar andbody mass, and adjusting the dosage upwards or downwards, as describedabove and known in the art.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention, should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side effects. Generally, treatment is initiated with smallerdosages, which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. In one embodiment of theinvention, the dosage range is 0.001% to 10% w/v. In another embodiment,the dosage range is 0.1% to 5% w/v.

However, typical doses may contain from a lower limit of about 0.1 mg toan upper limit of about 200 mg of the pharmaceutical compound per day.Also contemplated are other dose ranges such as 1 mg to 100 mg of thecompound per dose, and 3 mg to 70 mg per dose. Typically, the dose ofengineered polypeptides with long duration of action is administered,for example, daily and even once weekly. The doses per day may bedelivered in discrete unit doses, provided continuously in a 24 hourperiod or any portion of that the 24 hours.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

The surprising dose-sparing property of the engineered polypeptidesdescribed herein, along with their surprisingly long plasma half-lifeand duration of pharmacological action, provides for a superiorpharmaceutical agent. The superior properties including dose-sparing,allow for lower dosing, thus less or less severe side-effects andimproved cost of goods, and/or more cost-effective and simplerformulations for once daily or once weekly administration not currentlyachieved by the parent compounds alone.

D. Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.1, p.1, 1975. Theexact formulation, route of administration, and dosage can be chosen bythe individual physician in view of the patient's condition and theparticular method in which the compound is used.

Without wishing to be bound by any theory, it is believed thatconjugation of an ABD albumin binding domain with a hormone domain asdescribed herein, can provide decreased immunogenicity as judged by areduction in immune response relative to the hormone domain without ABDconjugation. See e.g., WO 2009/016043, incorporated herein by referencein its entirety and for all purposes.

VII. Examples

Examples 1-5 are provided to illustrate, amongst other things, thesuperior properties of the ABDs described herein, e.g. reducedimmunogenicity properties, compared to previous ABDs.

Example 1 Cloning, Expression, Purification and Characterization ofAlbumin Binding Polypeptides

In this example, eight different albumin binding polypeptides, PEP07913(SEQ ID NO:453), PEP07912 (SEQ ID NO:456), PEP07914 (SEQ ID NO:458),PEP07968 (i.e. DOTA conjugated to PEP07911, SEQ ID NO:459), PEP06923(SEQ ID NO:454), PEP07271 (SEQ ID NO:455), PEP07554 (SEQ ID NO:456) andPEP07844 (SEQ ID NO:461), the amino acid sequences of which are set outin FIG. 1, were cloned, purified and characterized.

Material and Methods Cloning of Albumin Binding Polypeptide Variants

After constructs were verified by sequencing, the vector DNA waspurified and transformed into an expression host, typically BL21(DE3). Asingle colony was selected to grow a starter culture in 4 ml LB mediafor ˜6 hrs.

Mutations in G148-GA3 were generated using site directed mutagenesiswith the appropriate oligonucleotides to obtain the desired albuminbinding polypeptide variants. The gene fragments were amplified by PCRwith primers adding specific endonuclease sites as well as an N-terminalMGSS (SEQ ID NO: 36) sequence preceding the albumin binding polypeptidevariants. The fragments were cleaved with NdeI and NotI, purified andligated to a cloning vector, the plasmid pAY02556 (containing an originof replication from pBR322, a kanamycin resistance gene and a T7promoter for expression of the gene of interest), restricted with thesame enzymes. Ligations were transformed to electrocompetent E. coliTOP10 cells. The transformed cells were spread on TBAB plates (30 g/ltryptose blood agar base) supplemented with 50 μg/ml of kanamycin,followed by incubation at 37° C. overnight. The colonies were screenedusing PCR and sequencing of amplified fragments was performed using thebiotinylated oligonucleotide and a BigDye® Terminator v3.1 CycleSequencing Kit (Applied Biosystems), used in accordance with themanufacturer's protocol. The sequencing reactions were purified bybinding to magnetic streptavidin coated beads using a Magnatrix 8000(NorDiag AB), and analyzed on ABI PRISM® 3100 Genetic Analyzer (PEApplied Biosystems). All albumin binding polypeptide variants weresubcloned as monomers and the constructs encoded by the expressionvectors were MGSS-[PP###] (“MGSS” disclosed as SEQ ID NO: 36), wherePP### corresponds to the 46 amino acid residues constituting thesequence of the albumin binding polypeptide.

In addition, the gene fragments of G148-GA3, PP007 (SEQ ID NO:307),PP013 (SEQ ID NO:313) and PP037 (SEQ ID NO:337) were amplified by PCRwith primers adding specific endonuclease sites as well as ahexahistidin (SEQ ID NO: 34) sequence, a TEV protease site and a glycineresidue before the 46 amino acid residues constituting the sequence ofthe albumin binding polypeptide. The polypeptides PEP07913 (SEQ IDNO:453), PEP07912 (SEQ ID NO:457), PEP07914 (SEQ ID NO:458) and PEP07968(SEQ ID NO:459) correspond to the albumin binding polypeptides G148-GA3,PP007 (SEQ ID NO:307), PP013 (SEQ ID NO:313) and PP037 (SEQ ID NO:337)with glycine residues added. The fragments were cleaved with XbaI andNotI, purified and ligated to a cloning vector, the plasmid pAY02512(containing an origin of replication from pBR322, a kanamycin resistancegene and a T7 promoter for expression of the gene of interest. Thecloning site is preceded by a sequence encoding a peptide containing ahexahistidine tag (SEQ ID NO: 34) followed by a cleavage site for theTEV protease), restricted with the same enzymes. Ligation,transformation and sequence verification were performed as describedabove. The four albumin binding polypeptide variants G148-GA3, PP007,PP013 and PP037 were subcloned as monomers and the constructs encoded bythe expression vectors were MGSSHHHHHHLQSSGVDLGTENLYFQG-[PP###], whereMGSSHHHHHHLQSSGVDLGTENLYFQG is SEQ ID NO:37.

Protein Expression

The albumin binding polypeptide variants were expressed in E. coli BL21(DE3) either with an N-terminal MGSS-extension (“MGSS” disclosed as SEQID NO: 36) or with an N-terminal His6-tag (SEQ ID NO: 34) followed by aTEV-protease recognition site and a glycine residue. A colony of eachalbumin binding polypeptide variant was used to inoculate 4 ml TSB+YEmedium supplemented with kanamycin to a concentration of 50 μg/ml. Thecultures were grown at 37° C. for approximately 5 hours. 3 ml from eachof the cultures was used to inoculate 800 ml TSB+YE supplemented withkanamycin to a concentration of 50 μg/ml in parallel bio reactors (Gretasystem, Belach Bioteknik AB). The cultivations were performed at 37° C.,with aeration at 800 ml/minute and an agitation profile to keepdissolved oxygen levels above 30%, to an OD600 of 2, which was followedby addition of IPTG to a final concentration of 0.5 mM. Cultivation wascontinued for five hours after which the cultivation was cooled to 10°C., aeration was stopped and agitation lowered to 300 rpm. Cell pelletswere harvested by centrifugation (15600×g, 4° C., 20 minutes) and storedat −20° C. until purification.

Purification of Albumin Binding Polypeptide Variants with a His6-Tag(SEQ ID NO: 34) and a TEV-Protease Site

Frozen cell pellets harboring soluble hexahistidine-tagged (SEQ ID NO:34) polypeptides PEP07913 (SEQ ID NO:453), PEP07912 (SEQ ID NO:456),PEP07914 (SEQ ID NO:458) and PEP07968 (SEQ ID NO:459) were suspended in35 ml binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mMimidazole, pH 7.4) with an addition of 1000 U Benzonase® (1.01654.001,Merck) and disrupted by ultrasonication. For each of the polypeptides,the ultrasonicated suspension was clarified by centrifugation (1 h,37000×g, 4° C.) and the supernatant was loaded onto a His GraviTrap™column (11-0033-99, GE Healthcare). The column was washed with 10 mlwashing buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH7.4), before eluting the polypeptide with 3 ml elution buffer (20 mMsodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4). The buffer wasexchanged to a cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8) usingPD-10 desalting column (17-0851-01, GE Healthcare). The amount ofpolypeptide product was determined by measuring the absorbance at 280 nmbefore adding DTT to a final concentration of 5 mM. His6-tagged (SEQ IDNO: 34) TEV protease was added to the cleavage buffer at a 1:10 massratio relative to the polypeptide product. The cleavage was performedover night under slow mixing at 4° C. Imidazole was added to thecleavage mix, to a concentration of 20 mM, before loading the mix onto aHis GraviTrap™ column (11-0033-99, GE Healthcare) for removing cleavedHis6-tags (SEQ ID NO: 34), His6-tagged (SEQ ID NO: 34) TEV protease andHis6-tagged (SEQ ID NO: 34) uncleaved product.

For each variant, the flow-through, containing the albumin bindingpolypeptide variant, was further purified by reversed phasechromatography (RPC), as follows. The flow-through fraction was loadedon 1 ml Resource 15 RPC column (GE Healthcare), previously equilibratedwith RPC A Buffer (0.1% TFA in water). After column wash with 10 columnvolumes (CV) RPC A Buffer, bound polypeptides were eluted with a lineargradient of 0-50% RPC B Buffer (0.1% TFA in acetonitrile) during 10 CV.The flow rate was 2 ml/min and the absorbance at 280 nm was monitored.Fractions containing albumin binding polypeptide variant were identifiedby SDS-PAGE analysis and pooled.

The RPC-purified albumin binding polypeptide variants were furtherpurified by gel filtration on 120 ml Superdex 75 (GE Healthcare) packedin an XK16 column (GE Healthcare). The running buffer was 1×PBS, and theflow rate 2 ml/min. Fractions containing pure albumin bindingpolypeptide variant were pooled and concentrated to approximately 1.3mg/ml. Finally, the concentrate was purified from trace amounts ofremaining endotoxins by using 1 ml columns of AffinityPak Detoxi-GelEndotoxin removing gel (Pierce, prod#20344), according to themanufacture's recommendations.

The albumin binding polypeptide variant PEP07911 was conjugated withMal-DOTA before the RPC-purification step, as follows. The buffer of theflow-through fraction from the IMAC-FT purification step was exchangedto 0.2 M NaAc, pH 5.5, using a disposable PD-10 desalting column (GEHealthcare). Maleimido-mono-amide-DOTA (Macrocyclics, cat. no. B-272)was added at 5-fold molar excess and incubated for 60 minutes at 30° C.under continuous shaking. The resulting polypeptide was denotedPEP07968.

Purification of Albumin Binding Polypeptide-Variants without His6-Tag(SEQ ID NO: 34)

Frozen cell pellets harboring soluble albumin binding polypeptidevariants PEP06923 (SEQ ID NO:454), PEP07271 (SEQ ID NO:455), PEP07554(SEQ ID NO:456) and PEP07844 (SEQ ID NO:461) were suspended in 20 mMTris-HCl, pH 8 and disrupted by ultrasonication. For each of thepolypeptide variants, the ultrasonicated suspension was clarified bycentrifugation (30 min, 32000×g, 4° C.) and the supernatant was loadedonto a HSA-Sepharose column (GE Healthcare). After washing withTST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH8.0), followed by 5 mM NH4Ac, pH 5.5, bound albumin binding polypeptidevariant was eluted with 0.5 M HAc, pH 3.2.

The albumin binding polypeptide variants were further purified byreversed phase chromatography (RPC), as follows. For each of thevariants, the eluate from the HSA-affinity purification step was loadedon 1 ml Resource 15 RPC column (GE Healthcare), previously equilibratedwith RPC A Buffer (0.1% TFA in water). After column wash with 10 CV RPCA Buffer, bound polypeptides were eluted with a linear gradient of 0-50%RPC B Buffer (0.1% TFA in acetonitrile) during 10 CV. The flow rate was2 ml/min and the absorbance at 280 nm was monitored. Fractionscontaining pure albumin binding polypeptide variants were identified bySDS-PAGE analysis and pooled. Finally, the buffer was exchanged to 1×PBS(2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) usinga disposable PD-10 desalting column (GE Healthcare).

Characterization of Purified Albumin Binding Polypeptide-Variants

The concentration was assessed by measuring the absorbance at 280 nmusing a NanoDrop® ND-1000 Spectrophotometer. The proteins were furtheranalyzed with SDS-PAGE and LC-MS.

For the SDS-PAGE analysis, approximately 10 μg of each albumin bindingpolypeptide variant was mixed with NuPAGE LDS Sample Buffer(Invitrogen), incubated at 70° C. for 15 min and loaded onto NuPAGE4-12% Bis-Tris Gels (Invitrogen). The gels were run with NuPAGE MES SDSRunning Buffer (Invitrogen) in an XCell II SureLock Electrophoresis Cell(Novex) employing the Sharp Prestained Standard (Invitrogen) asmolecular weight marker and using PhastGel BlueR (GE Healthcare) forstaining.

To verify the identity of the albumin binding polypeptide variants,LC/MS analyses were performed using an Agilent 1100 LC/MSD system,equipped with API-ESI and a single quadruple mass analyzer.Approximately 10 μg of each of the purified albumin binding polypeptidevariants was loaded on a Zorbax 300SB-C8 Narrow-Bore column (2.1×150 mm,3.5 μm, Agilent Technologies) at a flow-rate of 0.5 ml/min. Polypeptideswere eluted using a linear gradient of 10-70% solution B for 15 min at0.5 ml/min. The separation was performed at 30° C. The ion signal andthe absorbance at 280 and 220 nm were monitored. The molecular weightsof the purified albumin binding polypeptide variants were confirmed byMS.

Results

The expression levels of the albumin binding polypeptide variants were10-30 mg product/g cell pellet, as estimated from SDS-PAGE analysis.

For all variants, the purity, as determined by SDS-PAGE analysis,exceeded 95% and the LC/MS analysis verified the correct molecularweights. After purification, between 1 and 8 mg of pure polypeptide wasobtained for each of the eight albumin binding polypeptide variants.

Example 2 Affinity Determination for Albumin Binding Polypeptides

In this example, PEP06923 (SEQ ID NO:454), PEP07271 (SEQ ID NO:455),PEP07844 (SEQ ID NO:461), PEP07912 (SEQ ID NO:457), PEP07913 (SEQ IDNO:453), PEP07914 (SEQ ID NO:458) and PEP07968, (i.e. DOTA-conjugated toPEP07911, SEQ ID NO:459), synthesized or expressed and purified inExample 1 were characterized for affinity to human serum albumin (HSA)using a Biacore instrument. PEP07913 corresponds to the amino acidsequence of G148-GA3 with addition of a N-terminal glycine residue,whereas PEP07271, PEP07844, PEP07912, PEP07914 and PEP07968 correspondto the albumin binding polypeptides of PP001 (SEQ ID NO:301), PP043 (SEQID NO:343), PP007 (SEQ ID NO:307), PP013 (SEQ ID NO:313) and PP037 (SEQID NO:337) with different N-terminal amino acid additions.

Material and Methods

Biosensor analysis on a Biacore2000 instrument (GE Healthcare) wasperformed with HSA (Albucult®, Novozyme), immobilized by amine couplingonto the carboxylated dextran layer of the surfaces of CM-5 chips(research grade; Biacore) according to the manufacturer'srecommendations. Surface 1 of the chip was activated and deactivated andused as a reference cell (blank surface) during injections, whereassurface 2 comprised HSA immobilized to 731 resonance units (RU) andsurface 4 comprised HSA immobilized to 955 RU. The purified albuminbinding polypeptide variants were diluted in running buffer HBS-EP (GEHealthcare) to 2.5 nM, 10 nM and 40 nM, and injected at a constantflow-rate of 50 μl/min for 5 minutes, followed by injection of HBS-EPfor 60 minutes. The surfaces were regenerated with one injection of 25μl HCl, 10 mM. The affinity measurements were performed in two sets; inthe first set HBS-EP, PEP06923, PEP07271, PEP07912, PEP07913, PEP07914and PEP07968 were injected (chip surface 2), and in the second setHBS-EP, PEP06923, PEP07844, PEP07912 and PEP07914 were injected (chipsurface 4). PEP06923 was injected twice in each run as a control. Theresults were analyzed with a BiaEvaluation software (GE Healthcare).Curves of the blank surface were subtracted from the curves of theligand surfaces.

Results

The Biacore 2000 instrument has a technical limitation, hinderingmeasurements of very high affinity. Hence, the purpose of the Biacorestudy was not to determine the exact kinetic parameters of the albuminbinding polypeptide variants' affinity for HSA. However, the resultsprovide a quantitative estimation of the relative affinities of thesepolypeptides for albumin. After subtraction of reference surface andbuffer injection, curves were fitted to a 1:1 (Langmuir) binding modelusing BIAevaluation software with correction for mass transfer and withRUmax set as a local parameter. Curves are shown in FIG. 2. The relativeK_(D), k_(a) (k_(on)) and k_(d) (k_(off)) values were estimated and arepresented in the Tables below.

TABLE X1 Kinetic parameters (k_(a), k_(d) and K_(D)) of albumin bindingpolypeptides to HSA, 1st set k_(a) (Ms⁻¹) k_(d) (s⁻¹) K_(D) (M) PEP079135.7 × 10⁵ 9.3 × 10⁻⁴ 1.6 × 10⁻⁹ PEP06923 (1) 2.9 × 10⁷ 2.9 × 10⁻⁵ 9.9 ×10⁻¹³ PEP06923 (2) 2.6 × 10⁷ 2.8 × 10⁻⁵ 1.1 × 10⁻¹² PEP07271 3.9 × 10⁶2.9 × 10⁻⁵ 7.5 × 10⁻¹² PEP07912 4.6 × 10⁶ 2.8 × 10⁻⁵ 6.2 × 10⁻¹²PEP07914 3.5 × 10⁶ 2.5 × 10⁻⁵ 7.2 × 10⁻¹² PEP07968 3.0 × 10⁶ 2.7 × 10⁻⁵9.0 × 10⁻¹²

TABLE X2 Kinetic parameters (ka, kd and KD) of albumin bindingpolypeptides to HSA, 2nd set k_(a) (Ms⁻¹) k_(d) (s⁻¹) K_(D) (M) PEP06923(1) 2.0 × 10⁷ 2.6 × 10⁻⁵ 1.3 × 10⁻¹² PEP06923 (2) 2.1 × 10⁷ 2.5 × 10⁻⁵1.2 × 10⁻¹² PEP07912 5.4 × 10⁶ 2.8 × 10⁻⁵ 5.2 × 10⁻¹² PEP07914 3.8 × 10⁶2.6 × 10⁻⁵ 6.9 × 10⁻¹² PEP07844 5.4 × 10⁶ 2.3 × 10⁻⁵ 4.4 × 10⁻¹²

As shown in Table X1 and X2, PEP07271 (SEQ ID NO:455), PEP07844 (SEQ IDNO:461), PEP07912 (SEQ ID NO:457), PEP07914 (SEQ ID NO:458) and PEP07968(PEP07911 conjugated with DOTA, SEQ ID NO:459) all seem to haveapproximately the same affinity for HSA, widely exceeding the affinityof the parent G148-GA3 (PEP07913; SEQ ID NO:453). The HSA affinity ofthese polypeptides is slightly lower compared to PEP06923 (SEQ IDNO:454), despite similar off-rate.

Example 3 Determination of Melting Temperature (Tm) for Albumin BindingPolypeptides

In this example, the albumin binding polypeptide variants PEP07913 (SEQID NO:453), PEP06923 (SEQ ID NO:454), PEP07271 (SEQ ID NO:455), PEP07554(SEQ ID NO:456), PEP07912 (SEQ ID NO:457), PEP07914 (SEQ ID NO:458),PEP07968 (PEP07911 conjugated with DOTA, SEQ ID NO:459) and PEP07844(SEQ ID NO:461), expressed and purified as described in Example 1, andthe albumin polypeptide variant PEP07975 (i.e. DOTA-conjugated PEP07834,SEQ ID NO:460, via Cys14 with maleimido-mono-amide-DOTA (Macrocyclics,Cat. No. B-272), were analyzed by CD analysis. PEP07913 corresponds tothe sequence of G148-GA3 having an N-terminal glycine residue, PEP06923is an engineered high affinity derivative previously described byJonsson et al, supra, whereas PEP07271, PEP07554, PEP07912, PEP07914,PEP07968, PEP07844 and PEP07975 are examples of the 46 amino acidresidues albumin binding polypeptides of PP001 (SEQ ID NO:301), PP007(SEQ ID NO:307), PP013 (SEQ ID NO:313), PP037 (SEQ ID NO:337) and PP043(SEQ ID NO:343) having different N-terminal amino acid additionsaccording to the present disclosure.

Material and Methods

Purified albumin binding polypeptide variants were diluted in 1×PBS, tofinal concentrations between 0.4 and 0.5 mg/ml. Circular dichroism (CD)analysis was performed on a Jasco J-810 spectropolarimeter in a cellwith an optical path-length of 1 mm. In the variable temperaturemeasurements, the absorbance was measured at 221 nm from 20° to 90° C.,with a temperature slope of 5° C./min.

Results

The melting temperatures (Tm) of the different albumin bindingpolypeptide variants were calculated by determining the midpoint of thetransition in the CD vs. temperature plot. The results are summarized inTable X3 below.

TABLE X3 Determined Tm values of tested albumin binding polypeptidevariants N-terminal Variant SEQ ID NO: # sequence³ Tm (° C.) PEP07913SEQ ID NO: 453 GL 61 PEP06923 SEQ ID NO: 454 GSSL 57 PEP07271 SEQ ID NO:455 GSSL 65 PEP07554 SEQ ID NO: 456 GSSL 58 PEP07912 SEQ ID NO: 457 GL53 PEP07914 SEQ ID NO: 458 GL 59 PEP07968 SEQ ID NO: 459¹ GL 53 PEP07975SEQ ID NO: 460^(1, 2) AL 50 PEP07844 SEQ ID NO: 461 GSSL 65 ¹The peptideis conjugated with maleimide-DOTA at the cysteine ²The peptide isamidated at the C-terminus ³Leucine (underlined) is the residue inposition 1 of the 46 amino acid sequence of the albumin bindingpolypeptide as defined in the first aspect of the present disclosure“GSSL” in Table X3 disclosed as SEQ ID NO: 38.

The polypeptide PEP07968 is identical to PEP07912, except for the formerhaving a cysteine residue in position 14, which is conjugated withmaleimide DOTA, and the latter a serine residue. Thus, the DOTAmodification should not affect the melting temperature. Also PEP07975 ismaleiamide-conjugated with DOTA using Cys14, and is identical toPEP07968 except for the C-terminal amide (resulting from the peptidesynthesis) and for having an N-terminal alanine instead of a glycine.Furthermore, comparing PEP07912 and PEP07554 reveals that an N-terminalserine gives a higher melting temperature than a glycine in the sameposition (5° C. difference in Tm). Thus, all albumin binding polypeptidevariants according to the present disclosure show Tm above 55° C.,except PEP07912 and DOTA-conjugated variants. Taking into considerationthe importance of the N-terminal portion, all the tested albumin bindingpolypeptides are superior to the derivative of Jonsson et al, e.g.PEP06923.

Example 4 Serum Response Analysis

The percentage of human serum containing IgG, capable of binding to aset of albumin binding polypeptides as disclosed herein was analyzed byELISA. In total, 149 serum samples corresponding to 127 individuals werescreened.

Material and Methods

ELISA plates (96-well, half area plates (Costar, cat. No. 3690)) werecoated with 50 μl/well of Albucult® (Novozyme) diluted to 8 μg/ml incoating buffer (Sigma, cat. No. 3041). The plates were coated over nightfor three days at 4° C. On the day of analysis, the plates were washedtwice with tap water and blocked for 2 hours with 100 μl of phosphatebuffered saline (PBS) containing 0.05% casein (PBSC). The plates wereemptied and 50 μl/well of the albumin binding polypeptides PEP07913 (SEQID NO:453), PEP06923 (SEQ ID NO:454), PEP07271 (SEQ ID NO:455), PEP07912(SEQ ID NO:457), PEP07554 (SEQ ID NO:456), PEP07914 (SEQ ID NO:458),PEP07968 (DOTA conjugated PEP07911, SEQ ID NO:459) and PEP07844 (SEQ IDNO:461), diluted to 2 μg/ml in PBSC were added according to a pre-madeplate layout. After incubation for two hours at room temperature (RT),the plates were washed in PBSC four times using an automated ELISAwasher. The 149 serum samples from 129 individuals were diluted 50 timesin PBSC by adding 24 μl serum to 1174 μl PBSC. 50 μl of the diluted serawas added per well according to the pre-made plate layout. Each serumsample was tested as a singlet. Positive and negative controls wereincluded on each plate and for each albumin binding polypeptide. Albuminbinding antibodies (50 μl, 0.5 μl/ml immunoglobulin solution prepared inhouse from sera from primates immunized with PEP06923) was added as apositive control and 50 μl PBSC was used as a negative control. Theplates were incubated for one hour at RT and subsequently washed fourtimes in PBSC using an automated ELISA washer. The bound IgG wasdetected with 50 μl/well of anti-human IgG (Southern Biotech, cat no2040-05) diluted 10 000 times in PBSC. After washing four times in PBSCusing an automated ELISA washer, 50 μl/well of substrate was added(Pierce cat. No. 34021). The reaction was stopped after 10-15 minutes bythe addition of 50 μl H2SO4 to each well, prior to measuring theabsorbance using a multi-well plate reader (Victor3, Perkin Elmer).

Results

Of the 149 sera screened for IgG binding to the albumin bindingpolypeptides, 23 were negative for all eight polypeptides(OD-value<0.1), i.e. showed no IgG bound to the polypeptides. Theanalysis was performed with the 126 sera that were positive for one ormore albumin binding polypeptides. The average absorbance was calculated(FIG. 3A) and the percentage of sera with OD-values values either <0.15(FIG. 3B) or >1.0 (FIG. 3C). The highest average OD-value and thehighest percentage of serum with IgG binding were obtained with PEP07913(SEQ ID NO:453), PEP06923 (SEQ ID NO:454) and PEP07844 (SEQ ID NO:461),whereas least reactivity was found against PEP07968 (DOTA conjugatedPEP07911, SEQ ID NO:459), PEP07914 (SEQ ID NO:458) and PEP07954 (SEQ IDNO:456).

Thus, the most reactive albumin binding polypeptides were the parentalG148-GA3 (PEP07913, SEQ ID NO:453) and the previously affinity improvedderivative (PEP06923, SEQ ID NO:454), having helix 1 retained fromG148-GA3. The third of the more reactive polypeptides (PEP07844, SEQ IDNO:461) contains the original lysine in position 14 in helix 1. Thisresidue is intended for conjugation, and will therefore not be exposedwhen used as such. The identical albumin binding polypeptide variant,except for having an alanine in position 14 (PEP07554, SEQ ID NO:456),is one of the least reactive.

Example 5 Immunogenicity Testing of Albumin Binding Polypeptides

PEP07913 (SEQ ID NO:453), PEP07912 (SEQ ID NO:457), PEP07914 (SEQ IDNO:458), and PEP07968 (i.e. DOTA conjugated PEP07911, SEQ ID NO:459)were screened for their ability to induce T cell proliferation inperipheral blood mononuclear cells (PBMC) from 52 human Caucasianindividuals (obtained from CRI-Labo Medische Analyse, Gent, Belgium).PEP07913 corresponds to the sequence of G148-GA3 having an N-terminalglycine residue, whereas PEP07912, PEP07914 and PEP07968, are examplesof the 46 amino acid residues albumin binding polypeptides of PP007 (SEQID NO:307), PP013 (SEQ ID NO:313) and PP037 (SEQ ID NO:337) havingdifferent N-terminal amino acid additions according to the presentdisclosure.

Materials and Methods

PBMCs, prepared according to standard cell biological methods, wereadded to a tissue culture (TC) treated 96-well round bottom plate(Falcon) in an amount of 300 000 PBMCs/well. The cells were stimulatedby addition of 100 μl/well of albumin binding polypeptides PEP07913,PEP07912, PEP07914 and PEP07968 in AIMV medium (Invitrogen) additionallycontaining 900 μg/ml (3-fold molar excess) of recombinant human albumin(Albucult®, Novozyme). This corresponded to a final concentration ofalbumin binding polypeptide of 30 μg/ml. The stimulation was done ineight-plicates, i.e. the same albumin binding polypeptide were added toeight wells in identical amounts and under the same conditions. Inpositive control wells, the cells were stimulated with either 30 μg/mlKeyhole Limpet Hemocyanin (KLH, Calbiochem) or 30 μg/ml tetanus toxoid(TT, Statens Serum Institute). In negative control wells, only AIMVmedium with or without 900 μg/ml of albumin were added.

Cell proliferation was assessed after seven days of culturing usingAlexa Fluor 488 Click-iT EdU flow cytometry assay kit (Invitrogen). 1μM/well of EdU incorporation marker was added on day six. On day seven,cells were washed, dissociated from the plate, washed again and stainedfor 30 minutes with anti-CD3-PerCP reagent (Becton Dickinson) andanti-CD4-Alexa647 reagent (Becton Dickinson). Following staining, thecells were washed, fixed (BD cellfix, BD biosciences), permeabilized(using saponin) and stained for EdU by addition of Click-iT reagentaccording to the manufacturer's protocol (Invitrogen). After completedstaining, cells were washed again and analyzed using flow cytometry(FACSCantoII, BD Biosciences). To assess the number of proliferatingcells, a fixed number of fluospheres (Invitrogen) was added to each wellbefore analysis. All staining procedures and washes were performeddirectly in the 96-well plate.

The raw FACSCantoII data were gated hierarchically on CD3+ CD4+ T cellsand the number of gated cells as well as their fluorescence intensity ofEdU-Alexa Flour 488 incorporation marker were recorded. The mean valuesof the number of proliferating cells/eight-plicate of protein treatedwells were compared to the positive and negative controls and theresulting ratios, described as stimulation indices (SI), werecalculated. Based on the SI and the variation between replicates,threshold SI-values were set to 2.0 and 0.5 for stimulation andinhibition, respectively.

Results

The albumin binding polypeptides PEP07913, PEP07912, PEP07914 andPEP07968 were assessed for their immunogenic potential in the presenceof 3-fold excess of recombinant human albumin in a target humanpopulation using an in vitro PBMC proliferation assay. Compared to thealbumin control, PEP07913 induced CD3+ CD4+ T cells proliferation in 6of 52 donors, PEP07912 in 5 of 52 donors and PEP07914 and PEP07968 in 1of 52 donors (FIG. 4A).

The mean stimulation index (SI) for all 52 donors was not significantlydifferent for PEP07914 and PEP07968 compared to the negative controlcontaining recombinant human albumin (p=0.79 and 0.48 respectively, FIG.4B). The SI for PEP07913 was significantly higher (p=0.002) whereas theSI for PEP07912 was higher but not significant (p=0.03, FIG. 4B).

As compared to buffer only, the number of responding individuals was 10for PEP07912, 7 for PEP07912, 2 for PEP07914, 1 for PEP07968, 2 forrecombinant human albumin, and 49 and 51 for the two positive controlsTT and KLH, respectively (FIG. 4C). The albumin binding polypeptideswere ranked according to their immunogenicity in the following order:PEP07913>PEP07912>PEP07914>PEP07968. Both PEP07914 and PEP07968 weredefined as non-immunogenic. The above results thus demonstrate that theimmunogenic potential of the albumin binding polypeptides of the presentdisclosure is low, as compared to the positive controls.

Example 6 Leptin In Vitro Functional Activity

Method. This assay measures receptor signaling following treatment ofcells expressing a modified Leptin receptor. Test samples were assumedat 100% purity and re-solvated to 10× assay concentration in stimulationbuffer. A total of 90 ul of each 10× compound was transferred into adeep well pp plate and serially diluted (3-fold series) with Stimulationbuffer using the Perkin Elmer Multiprobe® II. The serially diluted platewas compounded into the 96-well stimulation plate containing 2.5×10̂5cell pellets of 18 hour leptin-weaned Keptin cells, as known in the art,using a MultiMek test program that transfers 200 ul of each of thediluted compounds and mixes the cells. At this time, the plate wassealed with an adhesive plate cover and placed at 37 C for 30 minutes toallow for stimulation of pSTAT5. See e.g., Crouse et al., 1998, J. Biol.Chem., 273:18365-18373. After incubation, the stimulation plate wascentrifuged to re-pellet the cells, the supernatant was removed and theremaining cell pellets were frozen at −80 C (>30 minutes). Cell lysateswere made by the addition of 100 ul of 1× lysate to the thawed cellpellets (Perkin Elmer pSTAT5 Assay kit) with rotation at ambient RT for20 minutes. The lysates were clarified at 2500 rpm for 20 minutes andexamined in the pSTAT5 Assay kit as 4 ul/well in a 384-well Proxiplate™according to manufacturer instructions. The pSTAT5 signal (RFU) wasdetermined using a Packard Fusion α-FP HT plate reader set to Alpha readparameters. Assay was completed in 384-well Proxiplate™ plates at 11 μltotal volume with values representing mean of n=4 assay wells per dosepoint.

Results

As set forth in Table X4 below, engineered polypeptides described hereinshow functional activity in the Obeca STAT5 assay.

TABLE X4 In vitro Functional Activity for Leptins Molecule or engineeredEC₅₀ nM (Obeca Cmpd polypeptide type STAT5 assay) 2 SEQ ID NO: 595 0.72567 SEQ ID NO: 660 1.38 12 SEQ ID NO: 605 0.947 1 SEQ ID NO: 594 0.908 68SEQ ID NO: 661 0.710 48 SEQ ID NO: 641 0.553 41 SEQ ID NO: 634 0.506 55SEQ ID NO: 648 0.639 69 SEQ ID NO: 662 0.832 70 SEQ ID NO: 663 0.941 63SEQ ID NO: 659 0.785 10 SEQ ID NO: 603 0.743 65 SEQ ID NO: 658 0.840 64SEQ ID NO: 657 0.634 9 SEQ ID NO: 602 0.433 71 SEQ ID NO: 681 1.258

Example 7 Leptin In Vitro Functional Activity in the Presence of Albumin

In this assay, the method described in Example 6 was used, exceptalbumin was added to the stimulation buffer to test leptin function ofCompound 2 in the presence of albumin. The albumins tested included 0.1%or 1% bovine serum albumin (BSA), 1% rat serum albumin (RSA), or 1%human serum albumin (HSA). The control sample was A100 leptin with 0.1%BSA.

Results.

As shown in FIG. 5, there were no effects of 1% Bovine/Rat/Human Albuminon the EC50 activity generated by Compound 2 in the Leptin FunctionAssay. The results are surprising and show that the therapeuticcompounds are active even when bound to albumin.

Example 8 Change in Body Weight and Food Intake after SingleAdministration of Engineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 315-330 grams at beginning of study. Animalswere divided into six groups (n=5/group). Each group was assigned toreceive one of the following: vehicle; Cmpd 2 at 13.33 nmol/kg; Cmpd 2at 40 nmol/kg; Cmpd 2 at 120 nmol/kg. Each test animal received a singlesubcutaneous injection at time=0. Food intake and change in body weight(% vehicle corrected) were monitored for 7 days, and the resultsrecorded as shown (FIGS. 6A and 6B). Administered compounds: Vehicle(circle); Cmpd 2 at 13.33 nmol/kg (triangle tip down); Cmpd 2 at 40nmol/kg (diamond); 120 nmol/kg (star).

Results.

As depicted in FIGS. 6A through 6B, administration of different doses ofthe engineered polypeptide resulted in reduced food intake and bodyweight relative to the group that received vehicle alone.

Example 9 Change in Body Weight and Food Intake after SingleAdministration of Engineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 315-330 grams at beginning of study. Eachtest animal (n=5/group) received a single subcutaneous injection attime=0. Animals were divided into five groups. Each group was assignedto receive one of the following: vehicle or Cmpd 66. Cmpd 66 wasdelivered at a dose of 120 nmol/kg. Food intake and change in bodyweight (% vehicle corrected) were monitored for 7 days, and the resultsrecorded as shown (FIGS. 7A and 7B). Administered compounds: Vehicle(circle); Cmpd 66 at 120 nmol/kg (triangle tip down).

Results.

As depicted in FIGS. 7A through 7B, administration of the engineeredpolypeptide resulted in reduced food intake and body weight relative tothe group that received vehicle alone.

Example 10 Change in Body Weight After Single Administration ofEngineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 315-330 g at beginning of study. Animalswere divided into six groups. Each test animal (n=5/group) received asingle subcutaneous injection at time=0. Each group was assigned toreceive one of the following: vehicle; Cmpd 2; or Cmpd 67. Cmpd 2 andCmpd 67 were each delivered at a dose of 120 nmol/kg. Percent change inbody weight for each group was monitored for 7 days, and the resultsrecorded as shown (FIG. 8). Administered compounds: Vehicle (circle);Cmpd 2 (triangle tip up); Cmpd 67 (diamond).

Results.

As depicted in FIG. 8, each group of animals that received a singleinjection of one of the engineered polypeptides tested exhibitedsignificant and sustained reduction in body weight relative to the groupthat received vehicle alone.

Example 11 Change in Body Weight After Single Administration ofEngineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 315-330 g at beginning of study. Animalswere divided into six groups. Each test animal (n=5/group) received asingle subcutaneous injection at time=0. Each group was assigned toreceive one of the following: vehicle; Cmpd 2; Cmpd 12; Cmpd 1. Cmpd 2,Cmpd 12, and Cmpd 1 were each delivered at a dose of 120 nmol/kg.Percent change in body weight for each group was monitored for 7 days,and the results recorded as shown (FIG. 9). Administered compounds:Vehicle (circle); Cmpd 2 (triangle tip up); Cmpd 12 (circle); Cmpd 1(triangle tip down).

Results.

As depicted in FIG. 9, each group of animals that received a singleinjection of one of the engineered polypeptides tested exhibitedsignificant and sustained reduction in body weight relative to the groupthat received vehicle alone.

Example 11 Affinity Determination for Albumin Binding Polypeptides

In this example, PEP07986 (SEQ ID NO:463), PEP07986-neutral linker-A500(Compound 2, SEQ ID NO:595), and PEP07986-negative linker-A500 (Compound12, SEQ ID NO:605) were characterized for affinity to different variantsof albumin.

Material and Methods

All studies were conducted on a BioRad ProteOn XPR36 system using a GLCsensor chip at 25 degrees C. For amine coupling the GLC chip wasactivated for 5 minutes using a 1:1 mixture of sulfo-NHS/EDC diluted30-fold from the initial stock in water as shown below. Each albuminsample was diluted to 25 ug/ml in 10 mM Na Acetate pH 5.0 and injectedfor 5 minutes over separate sensor surfaces. Each surface was thenblocked with 1 M ethanolamine pH 8.5. Each albumin was coupled at adensity of 2000-5000 in resonance units.

The binding of an engineered polypeptide was tested using 5 nM as thehighest concentration in a three-fold dilution series. The runningbuffer contained 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005%tween-20. All samples were tested using a 3-fold dilution series. Eachconcentration series was tested in duplicate. The dissociation phase forthe highest concentration highest concentration was monitored for 3hours.

Results

The relative K_(D) measured for the engineered polypeptides arepresented in Table X5 below. The results show that the albumin bindingpolypeptides associate with serum albumins (SA) with high affinity.

TABLE X5 K_(D) of albumin binding polypeptides to albumin variants HumanMonkey Dog Mouse Compound Name Rat SA SA SA SA SA Units PEP07986 6 9 84126 160 pM PEP07986- 23 152 405 1,310 1,660 Neutral-A500 PEP07986- 10270 400 1,810 1,940 Negative-A500

Example 12 Solubility of Engineered Polypeptides

As set forth in Table X6 following, engineered polypeptides describedherein have surprisingly high solubility in neutral pH.

Solubility was measured with the following assay: 6-10 mg of purifiedproteins were concentrated at 4° C. with centrifugal filter units(Amicon Ultra-15 or Ultra-4, with 3KDa MW cutoff; Millipore) to a volumeof less than 0.5 ml. They were centrifuged at 14,000 rpm for 10 minutesat 4° C. to remove precipitates and the supernatant was transferred to anew tube. The proteins were allowed to equilibrate overnight at roomtemperature in the dark, then were filtered with 0.22 micron syringefilters (Milex GV; Millipore) to remove precipitates. The absorbance atOD280 was measured with a NanoDrop spectrophotometer and theconcentration was calculated using the protein's theoretical molarextinction coefficient.

TABLE X6 Solubility of Engineered Polypeptides Solubility in PBS, pH 7.4Compound pI* Net Charge at pH 7.4* (mg/mL)** A100 6.2 −2.8 2.1 A-500 6.2−2.8 42.9 ABD2-A500 5.4 −5.8 16.8 (Cmpd 2) ABD2-HuSeal 9.4 +4.0 31 (Cmpd64)

Example 13 Stability of Engineered Polypeptides

Engineered polypeptides described herein are physically stable. Table X7shows the results of size exclusion chromatography (SEC) performed onA100 and ABD2-HuSeal. The engineered polypeptide show little to noself-association to dimer/oligomer, compared to A100.

TABLE X7 Stability of Engineered Polypeptides Compound Pk 1 (%) Pk 2 (%)Pk 3 (%) ABD2-HuSeal 98.78 1.22 n/a A100 88.21 11.15 0.65 Pk 1 = MonomerPk 2 = Dimer Pk 3 = Oligomer (Trimer/Tetramer)

SEC Method:

Column—Tosoh TSK Gel G3000 SWx1 7.8 mm×30 cm (#08541)

Mobile Phase—10 mM Na Phosphate, pH 7.4+238 mM NaCl+2.7 mM KCl RunTime—22 min

Flow Rate—0.8 mL/min

Column Temp—25° C. Sample Temp—5° C.

Sample load—40 ug

Detection—214 nm Example 14 Synergy of Amylin and Leptin is Absent inHigh BMI Subjects

Previous studies had described amylin/leptin synergy in rats weighing500-550 grams. After an inverse relationship of efficacy and BMI wasnoted we assessed the effects of the combination in very obese rats (750grams) and in very obese rats that were food restricted to themoderately obese range (500-550 g) range prior to initiating drugtreatment.

In this study one group of very obese rats (750 g) were allowed to feedad-libitum and were treated with amylin, leptin or the combination ofamylin+leptin. Although amylin was effective, there was no synergyevident with the addition of leptin. A second group of very obese rats(750 g) was calorie restricted down to the 500-550 g range in whichsynergy was previously demonstrated. These animals then beganamylin/leptin treatment and were allowed to feed ad-libitum. FIG. 10shows the results of the study. Rapid weight regain was evident invehicle and leptin monotherapy-treated rats. Some weight maintenance wasachieved with amylin monotherapy. No further weight maintenance wasachieved with the combination. These findings suggest that the lack ofsynergy in “high BMI” rodents cannot simply be rescued by a diet-leadin.

Example 15 Synergy of Engineered Polypeptides with Amylin Agonists

This study shows that a once weekly administration of ABD2-HuSeal(Compound 64) is sufficient for synergy when co-adminstered with a twiceweekly administration of PEG-rat amylin (Des-Lys1-[Lys26(mPEG40K)]-RatAmylin (SEQ ID NO: 148), Compound 124). FIG. 11 shows that thecombination of the engineered polypeptide and PEG-rat amylin resulted inmore weight loss than the results observed for each agent alone.ABD2-HuSeal was administered at 120 nmol/kg and PEG-amylin wasadministered at 125 nmol/kg to male DIO HSD rats of 500 g averageweight.

Example 16 Anti-Diabetic Effects of Engineered Polypeptides inNon-Obese, Type 1 Diabetic Mice

The purpose of this study was to evaluate the in vivo effects ofengineered polypeptides on key diabetic and metabolic endpoints in ahigh-dose STZ mouse model of Type 1 diabetes mellitus (T1DM). C57 BL/6male mice were given a single interperitoneal injection of STZ at 200mg/kg to induce Type 1 diabetes. Compounds were administered twice aweek subcutaneously for two weeks. Measured endpoints included HbA1clevels, glucose levels, body weight, and food intake.

FIG. 12 shows that Compound 2 produced a dose-related decrease in bloodglucose in STZ-induced diabetic mice. It also produced a dose-relateddecrease in Hemoglobin A1c levels, as shown in FIG. 13, and reduced bodyweight and cumulative food intake, as shown in FIG. 14.

In order to ensure that the glucose lowering effects of therapy are notdue to insulin effects, another study was conducted to combine theleptin therapy with a low dose of insulin. Compound 2 was administeredwith or without the addition of a 0.05 U/day dose of insulin in ahigh-dose STZ mouse model of T1DM. C57 BL/6 male mice were given asingle interperitoneal injection of STZ at 175 mg/kg to induce Type 1diabetes. Compounds were administered twice a week subcutaneously at 60nmol/kg for two weeks. Measured endpoints included HbA1c levels, glucoselevels, body weight, and food intake.

FIG. 15 shows a glucose lowering effect potentiated with low dose ofinsulin in an additive fashion for Compound 2. It also reducedHemoglobin A1c levels, as shown in FIG. 16, and reduced body weight andcumulative food intake, as shown in FIG. 17.

VIII. Embodiments

Additional embodiments of the engineered polypeptides, method of usethereof, and pharmaceuticals compositions described herein follow:

Embodiment 1

An engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD) and a first peptide hormone domain (HD1) selected froma leptin, a leptin analog or an active fragment thereof,

-   -   wherein said ABD comprises an amino acid sequence selected from        the amino acid sequence comprising:

(SEQ ID NO: 300) (i) LA X3 AK X6 X7 AN X10 ELD X14 YGVSDF YKRLI X26 KAKTVEGVEALK X39 X40 IL X43 X44 LPwherein independently of each otherX3 is selected from E, S, Q and C;X6 is selected from E, S and C;X7 is selected from A and S;X10 is selected from A, S and R;X14 is selected from A, S, C and K;X26 is selected from D and E;X39 is selected from D and E;X40 is selected from A and E;X43 is selected from A and K;X44 is selected from A, S and E;the leucine at position 45 is present or absent; andthe proline at position 46 is present or absent; and

-   -   (ii) an amino acid sequence which has at least 95% identity to        the sequence defined in (i);

with the proviso that X₇ is not L, E or D;

or alternatively,

with the proviso that the amino acid sequence is not defined by thefollowing sequence, as defined in PCT Published Application No. WO2009/016043: LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY GVSD X₅ YK X₈X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ ILAALP (SEQ ID NO: 679)

wherein

independently of each other,

X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K;

X_(e) is selected from D and K; and

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and

X₂₅ is selected from H, E and D.

Embodiment 2

The engineered polypeptide according to embodiment 1, further comprisinga first linker (L1) covalently linked to said HD1.

Embodiment 3

The engineered polypeptide according to embodiment 1 or 2, wherein saidengineered polypeptide comprises said ABD as an N-terminal moiety andsaid HD1 as a C-terminal moiety.

Embodiment 4

The engineered polypeptide according to embodiment 1 or 2, wherein saidengineered polypeptide comprises said ABD as a C-terminal moiety andsaid HD1 as an N-terminal moiety.

Embodiment 5

The engineered polypeptide according to embodiment 3, comprising thestructure: ABD-HD1.

Embodiment 6

The engineered polypeptide according to embodiment 3, comprising thestructure: ABD-L1-HD1.

Embodiment 7

The engineered polypeptide according to embodiment 4, comprising thestructure: HD1-ABD.

Embodiment 8

The engineered polypeptide according to embodiment 4, comprising thestructure: HD1-L1-ABD.

Embodiment 9

The engineered polypeptide according to any one of embodiments 1 to 8,wherein said HD1 is said a leptin, a leptin analog, a leptin activefragment, or a leptin derivative.

Embodiment 10

The engineered polypeptide according to any one of embodiments 1 to 9,wherein said HD1 has at least 50% identity with an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673,SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ IDNO:680.

Embodiment 11

The engineered polypeptide according to any one of embodiments 1 to 10,wherein said HD1 has at least 90% identity with an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673,SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ IDNO:680.

Embodiment 12

The engineered polypeptide according to any one of embodiments 1 to 11,wherein said HD1 has at least 50% identity with a human leptin.

Embodiment 13

The engineered polypeptide according to any one of embodiments 1 to 12,wherein said HD1 has at least 90% identity with a human leptin.

Embodiment 14

The engineered polypeptide according to any one of embodiments 1 to 13,wherein said HD1 has at least 50% identity with SEQ ID NO:20.

Embodiment 15

The engineered polypeptide according to any one of embodiments 1 to 14,wherein said HD1 has at least 90% identity with SEQ ID NO:20.

Embodiment 16

The engineered polypeptide according to any one of embodiments 1 to 15,wherein said HD1 has at least 50% identity with a platypus leptin.

Embodiment 17

The engineered polypeptide according to any one of embodiments 1 to 16,wherein said HD1 has at least 50% identity with a seal leptin.

Embodiment 18

The engineered polypeptide according to any one of embodiments 1 to 17,wherein said HD1 has from 1 to 5 amino acid modifications selectedindependently from any one or combination of an insertion, deletion,addition and substitution.

Embodiment 19

The engineered polypeptide according to any one of embodiments 1 to 18,wherein said HD1 comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ IDNO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ ID NO:665, SEQID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670,SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ IDNO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680.

Embodiment 20

The engineered polypeptide according to any one of embodiments 1 to 19,wherein said HD1 comprises an amino acid sequence that is selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680.

Embodiment 21

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:1.

Embodiment 22

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:2.

Embodiment 23

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:3.

Embodiment 24

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:4.

Embodiment 25

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is: SEQ ID NO:5.

Embodiment 26

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:6.

Embodiment 27

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:7.

Embodiment 28

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:8.

Embodiment 29

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:9.

Embodiment 30

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:10.

Embodiment 31

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:11.

Embodiment 32

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:12.

Embodiment 33

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:13.

Embodiment 34

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:14.

Embodiment 35

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:15.

Embodiment 36

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:16.

Embodiment 37

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:17.

Embodiment 38

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:18.

Embodiment 39

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:19.

Embodiment 40

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:20.

Embodiment 41

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:21.

Embodiment 42

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:22.

Embodiment 43

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:23.

Embodiment 44

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:24.

Embodiment 45

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:25.

Embodiment 46

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:26.

Embodiment 47

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:27.

Embodiment 48

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:28.

Embodiment 49

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:29.

Embodiment 50

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:30.

Embodiment 51

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:31.

Embodiment 52

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:32.

Embodiment 53

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:33.

Embodiment 54

The engineered polypeptide according to any one of embodiments 1 to 53,wherein said ABD comprises an amino acid sequence selected from theamino acid sequence comprising:

(SEQ ID NO: 300) (i) LA X3 AK X6 X7 AN X10 ELD X14 YGVSDF YKRLI X26 KAKTVEGVEALK X39 X40 IL X43 X44 LPwherein independently of each otherX3 is selected from E, S, Q and C;X6 is selected from E, S and C;X7 is selected from A and S;X10 is selected from A, S and R;X14 is selected from A, S, C and K;X26 is selected from D and E;X39 is selected from D and E;X40 is selected from A and E;X43 is selected from A and K;X44 is selected from A, S and E;the leucine at position 45 is present or absent; andthe proline at position 46 is present or absent.

Embodiment 55

The engineered polypeptide according to any one of embodiments 1 to 54,wherein said ABD comprises an amino acid sequence comprising: (ii) anamino acid sequence which has at least 95% identity to the sequencedefined in (i).

Embodiment 56

The engineered polypeptide according to any one of embodiments 1 to 55,wherein said ABD comprises an amino acid sequence selected from theamino acid sequence comprising:

(i) LA X3 AK X6 X7 AN X10 ELD X14 YGVSDFYKRLIDKAKTVEGVEALKDA ILAALP (SEQID NO: 678) wherein independently of each other X3 is selected from E,S, Q and C; X6 is selected from E, S and C; X7 is selected from A and S;X10 is selected from A, S and R; X14 is selected from A, S, C and K; theleucine at position 45 is present or absent; andthe proline at position 46 is present or absent; and(ii) an amino acid sequence which has at least 95% identity to thesequence defined in (i), with the proviso that X₇ is not L, E or D;or alternatively,with the proviso that the amino acid sequence is not defined by thefollowing sequence, as defined in PCT Published Application No. WO2009/016043: LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY GVSD X₅ YK X₈X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ ILAALP (SEQ ID NO: 679)wherein

independently of each other,

X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K;

X_(e) is selected from D and K; and

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and

X₂₅ is selected from H, E and D.

Embodiment 57

The engineered polypeptide according to any one of embodiments 1 to 56,wherein said ABD comprises an amino acid sequence selected from theamino acid sequence comprising:

(i) LA X3 AK X6 X7 AN X10 ELD X14 YGVSDFYKRLIDKAKTVEGVEALKDA ILAALP (SEQID NO: 678) wherein independently of each other X3 is selected from E,S, Q and C; X6 is selected from E, S and C; X7 is selected from A and S;X10 is selected from A, S and R; X14 is selected from A, S, C and K; theleucine at position 45 is present or absent; andthe proline at position 46 is present or absent.

Embodiment 58

The engineered polypeptide according to any one of embodiments 1 to 57,wherein said ABD comprises an amino acid sequence comprising: (ii) anamino acid sequence which has at least 95% identity to the sequencedefined in (i).

Embodiment 59

The engineered polypeptide according to any one of the precedingembodiments, wherein X6 is E in the ABD.

Embodiment 60

The engineered polypeptide according to any one of the precedingembodiments, wherein X3 is S in the ABD.

Embodiment 61

The engineered polypeptide according to any one of the precedingembodiments, wherein X3 is E in the ABD.

Embodiment 62

The engineered polypeptide according to any one of the precedingembodiments, wherein X7 is A in the ABD.

Embodiment 63

The engineered polypeptide according to any one of the precedingembodiments, wherein X14 is S in the ABD.

Embodiment 64

The engineered polypeptide according to any one of the precedingembodiments, wherein X14 is C in the ABD.

Embodiment 65

The engineered polypeptide according to any one of the precedingembodiments, wherein X10 is A in the ABD.

Embodiment 66

The engineered polypeptide according to any one of the precedingembodiments, wherein X10 is S in the ABD.

Embodiment 67

The engineered polypeptide according to any one of the precedingembodiments, wherein proline at position 46 is absent.

Embodiment 68

The engineered polypeptide according to any one of embodiments 56 to 58,wherein X26 is D in the ABD.

Embodiment 69

The engineered polypeptide according to any one of embodiments 56 to 58,wherein X39 is D in the ABD.

Embodiment 70

The engineered polypeptide according to any one of embodiments 56 to 58,wherein X40 is A in the ABD.

Embodiment 71

The engineered polypeptide according to any one of embodiments 56 to 58,wherein X43 is A in the ABD.

Embodiment 72

The engineered polypeptide according to any one of embodiments 56 to 58,wherein X44 is A in the ABD.

Embodiment 73

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD binds to albumin such that the koff valueof the interaction is at most 5×10⁻⁵ s⁻¹.

Embodiment 74

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD binds to albumin such the koff value of theinteraction is at most 5×10⁻⁶ s⁻¹.

Embodiment 75

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD is selected from any one of SEQ IDNO:301-452, 455-463, and 500-593.

Embodiment 76

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD is selected from an amino acid sequenceselected from any one of SEQ ID NO:313, SEQ ID NO:448, SEQ ID NO:463,SEQ ID NO:500, SEQ ID NO:501, and SEQ ID NO:502.

Embodiment 77

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD further comprising one or more additionalamino acid residues positioned at the N- and/or the C-terminal of thesequence defined in (i).

Embodiment 78

The engineered polypeptide according any one of the precedingembodiments, wherein the one or more additional amino acid residuescomprise a serine residue at the N-terminal of the ABD.

Embodiment 79

The engineered polypeptide according to any one of the precedingembodiments, wherein the one or more additional amino acid residuescomprise a glycine residue at the N-terminal of the ABD.

Embodiment 80

The engineered polypeptide according to any one of the precedingembodiments, wherein the one or more additional amino acid residuescomprise a cysteine residue at the N-terminal of the ABD.

Embodiment 81

The engineered polypeptide according to any one of the precedingembodiments, wherein the one or more additional amino acid residuescomprise a glycine residue at the C-terminal of the ABD.

Embodiment 82

The engineered polypeptide according to any one of the precedingembodiments, wherein the one or more additional amino acid residuescomprise a cysteine residue at the C-terminal of the ABD.

Embodiment 83

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD comprises an amino acid sequence selectedfrom any one of SEQ ID NO:445-450 and SEQ ID NO:462-463.

Embodiment 84

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD comprises no more than two cysteineresidues.

Embodiment 85

The engineered polypeptide according to any one of the precedingembodiments, wherein the ABD comprises no more than one cysteineresidue.

Embodiment 86

The engineered polypeptide according any one of the precedingembodiments, wherein the HD1 is conjugated to the ABD via a thiol groupof a cysteine residue at position X₁₄ of the polypeptide.

Embodiment 87

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 is a peptide of from 1 to 30 aminoacids or less than 30 amino acids.

Embodiment 88

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 is selected from the 20 naturallyoccurring amino acids.

Embodiment 89

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 is a non-natural amino acidsincorporated by chemical synthesis, post-translational chemicalmodification or by in vivo incorporation by recombinant expression in ahost cell.

Embodiment 90

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 amino acids are selected fromserine, glycine, alanine, proline, asparagine, glutamine, glutamate,aspartate, and lysine.

Embodiment 91

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a majority of amino acidsthat are sterically unhindered.

Embodiment 92

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises one or more of thefollowing: an acidic linker, a basic linker, and a structural motif

Embodiment 93

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises polyglycine, polyalanines,poly(Gly-Ala), or poly(Gly-Ser).

Embodiment 94

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a polyglycine of (Gly)₃,(Gly)₄ (SEQ ID NO: 116), or (Gly)₅ (SEQ ID NO: 117).

Embodiment 95

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises (Gly)₃Lys(Gly)₄ (SEQ IDNO: 118); (Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 119); (Gly)₃Cys(Gly)₄ (SEQID NO: 120); and GlyProAsnGlyGly (SEQ ID NO: 121).

Embodiment 96

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a combination of Gly andAla.

Embodiment 97

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a combination of Gly andSer.

Embodiment 98

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a combination of Gly andGlu.

Embodiment 99

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a combination of Gly andLys.

Embodiment 100

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a sequence selected fromthe group consisting of: [Gly-Ser]_(n) (SEQ ID NO: 122),[Gly-Gly-Ser]_(n) (SEQ ID NO: 123), [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO:124) and [Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 125); where n is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10.

Embodiment 101

The engineered polypeptide according any one of the precedingembodiments, wherein said linker L1 comprises a sequence selected fromthe group consisting of: [Gly-Glu]_(n) (SEQ ID NO: 126);[Gly-Gly-Glu]_(n) (SEQ ID NO: 127); [Gly-Gly-Gly-Glu]_(n) (SEQ ID NO:128); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 129), [Gly-Asp]_(n) (SEQ IDNO: 130); [Gly-Gly-Asp]_(n) (SEQ ID NO: 131); [Gly-Gly-Gly-Asp]_(n) (SEQID NO: 132); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 133) where n is 1, 2,3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 102

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a sequence selected fromthe group consisting of: [Gly-Glu]_(n) (SEQ ID NO: 126);[Gly-Gly-Glu]_(n) (SEQ ID NO: 127); [Gly-Gly-Gly-Glu]_(n) (SEQ ID NO:128); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 129), [Gly-Asp]_(n) (SEQ IDNO: 130); [Gly-Gly-Asp]_(n) (SEQ ID NO: 131); [Gly-Gly-Gly-Asp]_(n) (SEQID NO: 132); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 133) where n is 1, 2,3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 103

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a sequence selected fromthe group consisting of: [Gly-Lys]_(n) (SEQ ID NO: 134);[Gly-Gly-Lys]_(n) (SEQ ID NO: 135); [Gly-Gly-Gly-Lys]_(n) (SEQ ID NO:136); [Gly-Gly-Gly-Gly-Lys]_(n) (SEQ ID NO: 137), [Gly-Arg]_(n) (SEQ IDNO: 138); [Gly-Gly-Arg]_(n) (SEQ ID NO: 139); [Gly-Gly-Gly-Arg]_(n) (SEQID NO: 140); [Gly-Gly-Gly-Gly-Arg]_(n) (SEQ ID NO: 141) where n is 1, 2,3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 104

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a sequence selected fromthe group consisting of: [Glu-Ala-Ala-Ala-Lys]_(n) (SEQ ID NO: 142),where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 105

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a sequence selected fromthe group consisting of: [Gly-Gly-Glu]₆ (SEQ ID NO: 153) [Gly-Gly-Lys]₆(SEQ ID NO: 154). [Glu-Ala-Ala-Ala-Lys]₃ (SEQ ID NO: 155),[Glu-Ala-Ala-Ala-Lys]₄ (SEQ ID NO: 156), or [Glu-Ala-Ala-Ala-Lys]₅ (SEQID NO: 157).

Embodiment 106

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises an N-terminal TGdipeptide.

Embodiment 107

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises a C-terminal AS dipeptide.

Embodiment 108

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises an N-terminal TG dipeptideand a C-terminal AS dipeptide.

Embodiment 109

The engineered polypeptide according to any one of the precedingembodiments, wherein said linker L1 comprises an amino acids sequencethat is selected from the group consisting of TG-(GGGS)₁ (SEQ ID NO:215), TG-(GGGS)₂ (SEQ ID NO: 216), TG-(GGGS)₃ (SEQ ID NO: 217),TG-(GGGS)₄ (SEQ ID NO: 218), TG-(GGGS)₅ (SEQ ID NO: 219), (GGGS)₁-AS(SEQ ID NO: 220), (GGGS)₂-AS (SEQ ID NO: 221), (GGGS)₃-AS (SEQ ID NO:222), (GGGS)₄-AS (SEQ ID NO: 223), (GGGS)₅-AS (SEQ ID NO: 224),TG-(GGGS)₁-AS (SEQ ID NO: 225), TG-(GGGS)₂-AS (SEQ ID NO: 226),TG-(GGGS)₃-AS (SEQ ID NO: 227), TG-(GGGS)₄-AS (SEQ ID NO: 228), andTG-(GGGS)₅-AS (SEQ ID NO: 229).

Embodiment 110

The engineered polypeptide according to any one of the precedingembodiments, wherein said TG dipeptide TG and/or said dipeptide AS areabsent or are replaced by a pair of amino acids selected from T, A, S,and G.

Embodiment 111

The engineered polypeptide according to any one of the precedingembodiments, wherein said polypeptide further comprises one or moreadditional linkers.

Embodiment 112

The engineered polypeptide according to any one of the precedingembodiments, wherein said engineered polypeptide comprises an amino acidsequence selected from the group consisting of: SEQ ID NO: 594 to 663and 681.

Embodiment 113

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:594.

Embodiment 114

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:595.

Embodiment 115

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:596.

Embodiment 116

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:597.

Embodiment 117

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:598.

Embodiment 118

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:599.

Embodiment 119

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:600.

Embodiment 120

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:601.

Embodiment 121

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:602.

Embodiment 122

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:603.

Embodiment 123

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:604.

Embodiment 124

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:605.

Embodiment 125

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:606.

Embodiment 126

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:607.

Embodiment 127

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:608.

Embodiment 128

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:609.

Embodiment 129

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:610.

Embodiment 130

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:611.

Embodiment 131

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:612.

Embodiment 132

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:613.

Embodiment 133

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:614.

Embodiment 134

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:615.

Embodiment 135

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:616.

Embodiment 136

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:617.

Embodiment 137

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:618.

Embodiment 138

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:619.

Embodiment 139

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:620.

Embodiment 140

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:621.

Embodiment 141

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:622.

Embodiment 142

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:623.

Embodiment 143

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:624.

Embodiment 144

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:625.

Embodiment 145

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:626.

Embodiment 146

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:627.

Embodiment 147

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:628.

Embodiment 148

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:629.

Embodiment 149

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:630.

Embodiment 150

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:631.

Embodiment 151

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:632.

Embodiment 152

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:633.

Embodiment 153

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:634.

Embodiment 154

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:635.

Embodiment 155

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:636.

Embodiment 156

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:637.

Embodiment 157

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:638.

Embodiment 158

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:639.

Embodiment 159

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:640.

Embodiment 160

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:641.

Embodiment 161

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:642.

Embodiment 162

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:643.

Embodiment 163

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:644.

Embodiment 164

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:645.

Embodiment 165

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:646.

Embodiment 166

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:647.

Embodiment 167

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:648.

Embodiment 168

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:649.

Embodiment 169

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:650.

Embodiment 170

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:651.

Embodiment 171

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:652.

Embodiment 172

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:653.

Embodiment 173

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:654.

Embodiment 174

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:655.

Embodiment 175

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:656.

Embodiment 176

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:657.

Embodiment 177

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:658.

Embodiment 178

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:659.

Embodiment 179

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:660.

Embodiment 180

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:661.

Embodiment 181

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:662.

Embodiment 182

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:663.

Embodiment 183

The engineered polypeptide according to any one of embodiments 1 to 112,wherein said engineered polypeptide comprises the amino acid sequenceset out in SEQ ID NO:681.

Embodiment 184

The engineered polypeptide according to any one of embodiments 1 to 183,having affinity for serum albumin with a dissociation constant less thanabout 10⁻⁶ mol/L.

Embodiment 185

The engineered polypeptide according to any one of embodiments 1 to 184,having affinity for serum albumin with a dissociation constant less thanabout 10⁻⁹ mol/L.

Embodiment 186

The engineered polypeptide according to any one of embodiments 1 to 185,having affinity for serum albumin with a dissociation constant less thanabout 10⁻¹² mol/L.

Embodiment 187

The engineered polypeptide according to any one of embodiments 1 to 186,wherein the polypeptide has a duration of action of at least 1 day.

Embodiment 188

The engineered polypeptide according to any one of embodiments 1 to 187,wherein the polypeptide has a duration of action of at least 3 days.

Embodiment 189

The engineered polypeptide according to any one of embodiments 1 to 188,wherein the polypeptide has a duration of action of at least 5 days.

Embodiment 190

The engineered polypeptide according to any one of embodiments 1 to 189,wherein the polypeptide has a duration of action of at least 5 days in ahuman subject.

Embodiment 191

A method for treating a disease or disorder in a subject, comprisingadministering a engineered polypeptide according to any one ofembodiments 1 to 190 to a subject in need thereof in an amount effectiveto treat said disease or disorder.

Embodiment 192

The method according to embodiment 191, wherein said disease or disorderis disease or disorder can be lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes(including type I and type II), nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X andHuntington's Disease.

Embodiment 193

The method according to embodiment 191 or embodiment 192, wherein saiddisease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia,overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease,leptin deficiency, fatty liver disease or diabetes.

Embodiment 194

The method according to any one of embodiments 191 to 193, wherein saiddisease or disorder is type I diabetes or type II diabetes.

Embodiment 195

The method according to any one of embodiments 191 to 193, wherein saiddisease or disorder is obesity.

Embodiment 196

The method according to any one of embodiments 191 to 193, wherein saiddisease or disorder is lipodystrophy or leptin deficiency.

Embodiment 197

A pharmaceutical composition comprising an engineered polypeptideaccording to any one of embodiments 1 to 190 and a pharmaceuticallyacceptable excipient.

Embodiment 198

The pharmaceutical composition according to embodiment 197, wherein saidpharmaceutical composition is an injectable pharmaceutical composition.

Embodiment 199

The pharmaceutical composition according to any one of embodiments 197to 198, wherein said pharmaceutical composition is a sustained releaseor long lasting pharmaceutical composition.

Embodiment 200

The pharmaceutical composition according to any one of embodiments 197to 199, wherein said pharmaceutical composition is a once dailypharmaceutical composition.

Embodiment 201

The pharmaceutical composition according to any one of embodiments 197to 199, wherein said pharmaceutical composition is a once weeklypharmaceutical composition.

Embodiment 202

A pharmaceutical composition of any one of embodiments 197 to 201 fortreating a disease or disorder in a subject.

Embodiment 203

The pharmaceutical composition according to any one of embodiments 197to 202 wherein the disease or disorder is lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes(including type I and type II), nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X andHuntington's Disease.

Embodiment 204

The pharmaceutical composition of embodiment 202 or embodiment 203wherein said disease or disorder is lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes.

Embodiment 205

The method according to any one of embodiments 202 to 204, wherein saiddisease or disorder is type I diabetes or type II diabetes.

Embodiment 206

The method according to any one of embodiments 202 to 204, wherein saiddisease or disorder is obesity.

Embodiment 207

The method according to any one of embodiments 202 to 204, wherein saiddisease or disorder is lipodystrophy or leptin deficiency.

Embodiment 208

The engineered polypeptide according to any one of embodiments 1 to 18,wherein said HD1 is selected from the group consisting of:

-   -   (a) the amino acid sequence 1-146 of a leptin selected from the        group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ        ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ        ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,        SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID        NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,        SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID        NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,        SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID        NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ ID NO:665, SEQ ID        NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID        NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID        NO:674, SEQ ID NO:675, SEQ ID NO:676, and SEQ ID NO:677; in        which a different amino acid is substituted in one or more of        the following positions and retaining the same numbering (even        in the absence of a glutaminyl residue at position 28): 4, 32,        33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106,        107, 108, 111, 118, 136, 138, 142, and 145;    -   (b) the amino acid sequence of subpart (a) in which the        glutaminyl residue at position 28 is absent;    -   (c) the amino acid sequence of subparts (a) or (b) in which a        methionyl residue is added at the N-terminus;    -   (d) a leptin consisting of a fragment of the amino acid sequence        of (a), (b), or (c) selected from the group consisting of:        -   (i) amino acids 98-146;        -   (ii) amino acids 1-32;        -   (iii) amino acids 40-116;        -   (iv) amino acids 1-99 and 112-146;        -   (v) amino acids 1-99 and 112-146 in which one or more of            amino acids 100-111 is placed between amino acids 99 and            112;        -   (vi) the amino acid sequence of subpart (i) wherein one or            more of amino acids 100, 102, 105, 106, 107, 108, 111, 118,            136, 138, 142, and 145 is substituted with another amino            acid;        -   (vii) the amino acid sequence of subpart (ii) wherein one or            more of amino acids 4, 8 and 32 is substituted with another            amino acid;        -   (viii) the amino acid sequence of subpart (iii) wherein one            or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74,            77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 is            replaced with another amino acid;        -   (ix) the amino acid sequence of subpart (iv) wherein one or            more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64,            66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142,            and 145 is replaced with another amino acid; and        -   (x) the amino acid sequence of subpart (v) wherein one or            more of amino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77,            78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136,            138, 142, and 145 is replaced with another amino acid;        -   (xi) the amino acid sequence of any of subparts (i)-(x)            wherein a methionine has been added at the N-terminus;    -   (e) the amino acid sequence of any of subparts (a) through (d)        wherein said amino acid sequence is attached to a chemical        moiety;    -   (f) the amino acid sequence of subpart (e) wherein said chemical        moiety is a water soluble polymer moiety;    -   (g) the amino acid sequence of subpart (f) wherein said water        soluble polymer moiety is selected from the group consisting of:        polyethylene glycol, an ethylene glycol/propylene glycol        copolymer, a carboxymethylcellulose, a dextran, a polyvinyl        alcohol, a polyvinyl pyrolidone, a poly-1,3-dioxolane, a        poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a        polyaminoacid homopolymer, a polyaminoacid random copolymer, an        albumin, an Fc protein, a poly(n-vinyl pyrolidone)polyethylene        glycol, a propylene glycol homopolymer, a polypropylene        oxide/ethylene oxide copolymer, a polyoxyethylated polyol, a        polyvinyl alcohol, a polyethylene glycol propionadehyde, a        succinate, a styrene, a hydroxyethyl starch and;    -   (h) the amino acid sequence of subpart (g) wherein said water        soluble polymer moiety is a polyethylene glycol; and    -   (i) the amino acid sequence of subpart (g) wherein said water        soluble polymer is a polyamino acid-selected from the group        consisting of: an albumin, an antibody, an Fc protein, and a        polylysine moiety.

Embodiment 209

The engineered polypeptide according to any one of embodiments 1 to 18and 208, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinone or more amino acid substitutions have been made.

Embodiment 210

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinone amino acid substitution has been made.

Embodiment 211

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereintwo amino acid substitutions have been made.

Embodiment 212

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinthree amino acid substitutions have been made.

Embodiment 213

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinfour amino acid substitutions have been made.

Embodiment 214

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinfive amino acid substitutions have been made.

Embodiment 215

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinsix amino acid substitutions have been made.

Embodiment 216

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinseven amino acid substitutions have been made.

Embodiment 217

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereineight amino acid substitutions have been made.

Embodiment 218

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinnine amino acid substitutions have been made.

Embodiment 219

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; whereinten amino acid substitutions have been made.

Embodiment 220

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein11 amino acid substitutions have been made.

Embodiment 221

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein12 amino acid substitutions have been made.

Embodiment 222

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein13 amino acid substitutions have been made.

Embodiment 223

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein14 amino acid substitutions have been made.

Embodiment 224

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein15 amino acid substitutions have been made.

Embodiment 225

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein16 amino acid substitutions have been made.

Embodiment 226

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein17 amino acid substitutions have been made.

Embodiment 227

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein18 amino acid substitutions have been made.

Embodiment 228

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein19 amino acid substitutions have been made.

Embodiment 229

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein20 amino acid substitutions have been made.

Embodiment 230

The engineered polypeptide according to any one of embodiments 1 to 18and 209, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQ IDNO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, and SEQ ID NO:680; wherein21 amino acid substitutions have been made.

Embodiment 231

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:143.

Embodiment 232

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:144.

Embodiment 233

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:145.

Embodiment 234

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:146.

Embodiment 235

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:664.

Embodiment 236

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:665.

Embodiment 237

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:666.

Embodiment 238

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:667.

Embodiment 239

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:668.

Embodiment 240

The engineered polypeptide according to any one of embodiments s 1 to20, wherein said HD1 is SEQ ID NO:669.

Embodiment 241

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:670.

Embodiment 242

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:671.

Embodiment 243

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:672.

Embodiment 244

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:673.

Embodiment 245

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:674.

Embodiment 246

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:675.

Embodiment 247

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:676.

Embodiment 248

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:677.

Embodiment 249

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:680.

While the foregoing description discloses the present invention, withexamples provided for the purpose of illustration, it will be understoodthat the practice of the present invention encompasses all of the usualvariations, adaptations, or modifications as being within the scope ofthe claimed invention. Therefore, descriptions and examples should notbe construed as limiting the scope of the invention, which is delineatedby the appended claims.

1. An engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD) and a first peptide hormone domain (HD1) comprising aleptin, a leptin analog or an active fragment thereof, wherein said ABDcomprises at least 95% identity to SEQ ID NO:300, with the proviso thatX₇ is not L, E or D; or alternatively, with the proviso that the aminoacid sequence is not SEQ ID NO:679.
 2. The engineered polypeptideaccording to claim 1, further comprising a first linker (L1) covalentlylinked to said HD1. 3-18. (canceled)
 19. The engineered polypeptideaccording to claim 1, wherein said HD1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:664, SEQID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ NO:669, SEQID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674,SEQ ID NO:675, SEQ ID NO:676, SEQ NO:677, and SEQ ID NO:680. 20-74.(canceled)
 75. The engineered polypeptide according to claim 1, whereinthe ABD is selected from any one of SEQ ID NO:301-452, 455-463, and500-591. 76-111. (canceled)
 112. The engineered polypeptide according toclaim 1, wherein said engineered polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 594 to 663 and681. 113-183. (canceled)
 184. The engineered polypeptide according toclaim 1, having affinity for serum albumin with a dissociation constantless than about 10⁻⁶ mol/L. 185-190. (canceled)
 191. A method fortreating a disease or disorder in a subject, comprising administering anengineered polypeptide according to claim 1 to a subject in need thereofin an amount effective to treat said disease or disorder.
 192. Themethod according to claim 191, wherein said disease or disorder islipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity,hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fattyliver disease, diabetes, nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD) metabolic syndrome X orHuntington's Disease. 193-196. (canceled)
 197. A pharmaceuticalcomposition comprising an engineered polypeptide according to claim 1and a pharmaceutically acceptable excipient. 198-283. (canceled)
 284. Apolynucleotide encoding an engineered polypeptide according to claim 1.285. An expression vector comprising a polynucleotide according to claim284.
 286. A host cell comprising an expression vector according to claim285.
 287. A method for preparing the engineered polypeptide of claim 1,the method comprising expressing a polynucleotide encoding theengineered polypeptide or synthesizing the polypeptide by non-biologicalpeptide synthesis.